Method for activating t cells for cancer treatment

ABSTRACT

The present invention relates to a cancer-specific tumor antigen neoepitope represented by any one of SEQ ID NOs: 1 to 214, an antigen-presenting cell loaded with the neoepitope, and a method for activating T cells for cancer treatment using the antigen-presenting cell. An antigen-presenting cell, that is, a dendritic cell, loaded with a cancer-specific tumor antigen epitope provided in the present invention enables rapid and effective induction of differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, and the memory T cells thus activated can treat a cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

TECHNICAL FIELD

The present invention relates to a cancer-specific tumor antigen neoepitope, an antigen-presenting cell loaded with the neoepitope, and a method for activating T cells for cancer treatment using the antigen-presenting cell.

BACKGROUND ART

Gastric cancer is known as a malignancy with a high incidence in the world, especially in Asia. There have been many known causes of development of gastric cancer; however, gastric cancer may be typically classified into EBV-associated gastric cancer, which is caused by infection with Epstein-Barr virus (EBV), and gastric cancer cell antigen-associated gastric cancer, which is caused by accumulation of genetic mutations in gastrointestinal cells. For current treatment for gastric cancer, excision of cancerous tissue has long been known to be the most effective, and chemotherapy and radiation therapy are also performed. However, it appears that gastric cancer is a hard-to-cure disease when not found early. In addition, although clinical trials have been conducted through several biological agents (antibodies, small molecules), therapeutic agents with good clinical effects have not yet been reported.

Recently, cancer cell-specific targeted therapy using patient-derived autologous T cells has been studied in several institutions, and clinical trials have been conducted for lymphoma using chimeric antigen receptor (CAR) T cells in several institutions. As a result, due to good clinical effects and low side effects, such therapy has attracted much attention as a new field of anticancer therapy.

Use of patient-derived T cells decreases induction of immune responses which is the biggest side effect of cell therapeutic agents, and removes restrictions on the donor's HLA type. Thus, such T cells have been known as therapeutic agents which are effective and have no side effects. To date, CD8+ T cells, CD4+ T cells, NK cells, dendritic cells, and CAR T cells are known as types of cell therapeutic agents which are most widely used in the field of anticancer therapy. NK cells have cell-killing efficacy, and have several side effects due to not having antigen specificity. Dendritic cells are therapeutic agents belonging to the vaccine concept in that they have no function of directly killing cells, and are capable of delivering antigen specificity to T cells in the patient's body so that cancer cell specificity is imparted to T cells with high efficiency. In addition, CD4+ T cells play a role in helping other cells through antigen specificity, and CD8+ T cells are known to have the best antigen specificity and cell-killing effect.

However, most cell therapeutic agents, which have been used or developed to date, have limitations and thus have no clinical effect. Taking a look at the limitations, cancer cells, on their own, secrete substances that suppress immune responses in the human body, or do not present antigens necessary for production of antibodies against such cancer cells, thereby preventing an appropriate immune response from occurring.

Meanwhile, dendritic cells not only act as surveillants to detect antigens that come from the outside of the human body or are produced internally, but also quickly travel to the secondary lymphoid organs with such recognized and absorbed antigen, thereby acting as specialized antigen-presenting cells that present the antigens to immune cells, including T cells, which react with the antigens. Anti-cancer immunotherapeutic vaccines using dendritic cells have been developed through several methods, and may be largely divided into ex vivo generated dendritic cell vaccines and in vivo dendritic cell vaccines. The in vivo dendritic cell vaccine works in a manner of directly delivering an antigen to dendritic cells present in the body. In addition, a method using the ex vivo generated dendritic cell vaccine is in such a manner that dendritic cells are isolated from the patient's PBMCs and an antigen to be presented is delivered to the isolated dendritic cells, through which the dendritic cells are activated and then injected back into the patient so that the antigen is delivered from the injected dendritic cells to T cells. In the latter, ex vivo dendritic cell culture method and antigen delivery method are important, and currently used antigen presentation methods include transfection of DNA of an antigen to be presented using virus or nucleofection, or antigen delivery targeting dendritic cells in which an antigen is bound to an antibody targeting the dendritic cells.

Currently, the biggest problems in dendritic cell vaccines are that severe chronic inflammatory phenomena in the body and the Warburg effect are exhibited, and it is considered very difficult to achieve effective activation of anticancer immune cells under the cancer microenvironment in which immunosuppressive cytokines, immunosuppressive T cells, dendritic cells, and the like are present.

TECHNICAL PROBLEM

An object of the present invention is to provide an Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope, and a composition for activating T cells which comprises the same.

Another object of the present invention is to provide an antigen-presenting cell loaded with a neoepitope of the present invention, the antigen-presenting cell being capable of activating T cells for cancer treatment.

Yet another object of the present invention is to provide a T cell, activated by the antigen-presenting cell loaded with a neoepitope of the present invention.

Still yet another object of the present invention is to provide a method for activating T cells for cancer treatment.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other problems which are not mentioned will be clearly understood by those skilled in the art from the following description.

SOLUTION TO PROBLEM

According to an embodiment of the present invention, there is provided a cancer-specific tumor antigen epitope.

In the present invention, the “cancer-specific tumor antigen epitope” is derived from a mutant protein antigen which is present only in cancer cells and is not present in normal cells. In the present invention, the cancer-specific tumor antigen epitope includes at least one epitope recognized by T cell receptors; and such an epitope may preferably include neoepitopes of autologous cancer antigens that appear due to mutation of cancer genes in Epstein-Barr virus (EBV)-negative cancer.

In the present invention, the “neoepitope” refers to an epitope that is not present in a reference such as normal, non-cancerous cells or germline cells and is found in cancer cells. This includes, in particular, a situation where a corresponding epitope is found in normal, non-cancer cells or germline cells, but one or more mutations in cancer cells cause the epitope to be changed to a neoepitope. Regarding neoepitopes, it may be considered that the neoepitopes express random mutations in tumor cells that produce unique and tumor-specific antigens. Therefore, viewed from a different perspective, the neoepitopes may be identified considering the type (for example, deletion, insertion, transversion, transition, translocation) and effect (for example, non-sense, mis sense, frame shift, and the like) of mutation, which may serve as a first content filter through which silent and other non-relevant mutations are eliminated. In addition, it should be appreciated that neoepitope sequences can be defined as sequence stretches with relatively short length (for example, 7- to 11-mer), in which such stretches will include change(s) in the amino acid sequence. Most typically, the changed amino acid will be at or near the central amino acid position. For example, a typical neoepitope may have a structure of A4-N-A4, or A3-N-A5, or A2-N-A7, or A5-N-A3, or A7-N-A2, where A is a proteinogenic amino acid and N is a changed amino acid (relative to wild type or relative to matched normal). For example, neoepitope sequences as contemplated herein include sequence stretches with relatively short length (for example, 5- to 30-mer, more typically 7- to 11-mer, or 12- to 25-mer), in which such stretches include change(s) in the amino acid sequence. Thus, it should be appreciated that a single amino acid change may be presented in numerous neoepitope sequences that include the changed amino acid, depending on the position of the changed amino acid. Advantageously, such sequence variability allows for multiple choices of neoepitopes, and thus increases the number of potentially useful targets that can then be selected on the basis of one or more desirable traits (for example, highest affinity to the patient's HLA-type, highest structural stability, and the like). Most typically, such a neoepitope will be calculated to have a length of between 2 to 50 amino acids, more typically between 5 to 30 amino acids, and most typically between 9 to 15 amino acids, with a changed amino acid preferably centrally located or otherwise situated in such a manner as to improve its binding to MHC. For example, in a case where the epitope is to be presented by MHC-I complex, a neoepitope will be typically about 8 to 11 amino acids in length, while the neoepitope presented via MHC-II complex will typically have about 13 to 17 amino acids in length. As will be readily appreciated, since the position of the changed amino acid in the neoepitope may be other than central, the actual peptide sequence and with that actual topology of the neoepitope may vary considerably.

In the present invention, the neoepitope may exhibit binding affinity with at least one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, f32-microglobulin, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci so that T cells, preferably memory T cells, extracted from human blood can have efficacy. Among these, the neoepitope may include those exhibiting high binding affinity with at least one of the HLA types that are most expressed by Koreans, for example, HLA-A*2402, HLA-A*A0201, HLA-A*3303, HLA-A*1101, HLA-A*0206, HLA-A*3101, HLA-B*5101, HLA-B*4403, HLA-B*5401, HLA-B*5801, and HLA-B*3501, and preferably with HLA-A*2402 or HLA-A*A0201.

Preferably, in the present invention, the neoepitope has high binding affinity for HLA-A*2402 and may be a neoepitope represented by any one of SEQ ID NOs: 1 to 49; or has high binding affinity for HLA-A*0201 and may be a neoepitope represented by any one of SEQ ID NOs: 50 to 214.

Here, in the present invention, for a method of measuring neoepitope-HLA affinity, NetMHC 3.4 (URL: www.cbs.dtu.dk/services/NetMHC-3.4/) may be used to predict whether a neoepitope binds to a specific HLA allele. However, the present invention is not limited thereto.

In the present invention, the “HLA” or “human leukocyte antigen” refers to human gene that encodes a major histocompatibility complex (MHC) protein on the surface of cells that are responsible for regulation of the immune system. “HLA-I” or “HLA class I” refers to human MHC class I gene including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and β2-microglobulin loci. “HLA-II” or “HLA class II” refers to human MHC class II gene including HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci.

In the present invention, the cancer may be Epstein-Barr virus (EBV)-negative cancer, and may include, without limitation, any cancer species as long as it expresses a neoepitope represented by any one of SEQ ID NOs: 1 to 214 of the present invention. Thus, the type thereof is not particularly limited, and examples thereof may include colorectal cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, ovarian cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma, with gastric cancer being preferred.

According to another embodiment of the present invention, there is provided a nucleic acid molecule, encoding a cancer-specific tumor antigen epitope provided in the present invention, preferably, a neoepitope of Epstein-Barr virus (EBV)-negative cancer antigen represented by any one of SEQ ID NOs: 1 to 214.

The nucleic acid molecule of the present invention includes any nucleic acid molecule obtained by converting an amino acid sequence of a polypeptide provided in the present invention into a polynucleotide sequence as known to those skilled in the art. Thus, various polynucleotide sequences may be prepared due to open reading frame (ORF), all of which are also included in the nucleic acid molecule of the present invention.

According to yet another embodiment of the present invention, there is provided an expression vector, into which the isolated nucleic acid molecule provided in the present invention is inserted.

In the present invention, the “vector” is a nucleic acid molecule which is capable of transporting another nucleic acid linked thereto. One type of vector is a “plasmid,” which refers to circular double-stranded DNA into which an additional DNA segment can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector, where an additional DNA segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. In addition, certain vectors are capable of directing expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors.” In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form among vectors.

Specific examples of the expression vector in the present invention may be selected from, but are not limited to, the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; plant viruses. Any expression vector known, to those skilled in the art, as expression vectors can be used in the present invention, and the expression vector is selected depending on the nature of the target host cell. Introduction of a vector into a host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofectamine transfection, or electroporation. However, the present invention is not limited thereto, and those skilled in the art may adopt and use an introduction method appropriate for the expression vector and the host cell which are used. The vector may preferably contain at least one selection marker. However, the present invention is not limited thereto, and selection can be made using the vector that contains no selection marker, depending on whether or not a product is produced. The selection marker is selected depending on the target host cell, which is done using methods already known to those skilled in the art, and thus the present invention has no limitation thereon.

In order to facilitate purification of the nucleic acid molecule of the present invention, a tag sequence may be inserted into and fused to an expression vector. The tag includes, but is not limited to, hexa-histidine tag, hemagglutinin tag, myc tag, or flag tag, and any tag known to those skilled in the art which facilitates purification can be used in the present invention.

According to still yet another embodiment of the present invention, there is provided a host cell, transfected with the expression vector provided in the present invention.

In the present invention, the “host cell” includes individual cells or cell cultures which may be or have been recipients of the vector(s) for incorporation of a polypeptide insert. The host cell includes progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) herein.

In the present invention, the host cell may include cells of mammalian, plant, insect, fungal, or cellular origin, and may be, for example, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as Chinese hamster ovary (CHO) cells, SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, or PERC.6 (human retinal cells); or plant cells. However, the host cell is not limited thereto, and any cell known to those skilled in the art which can be used as a host cell is available.

According to still yet another embodiment of the present invention, there is provided a composition for activating T cells, comprising a cancer-specific tumor antigen epitope provided in the present invention, a nucleic acid molecule encoding the same, an expression vector into which the nucleic acid molecule is inserted, or a host cell transformed with the expression vector.

As used herein, the term “activation of T cells” refers to a population of monoclonal (for example, encoding the same TCR) or polyclonal (for example, having clones encoding different TCRs) T cells that have T cell receptors recognizing at least one tumor antigen peptide. Activated T cells may include one or more subtypes of T cells, including, but not limited to, one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells, with memory T cells being preferred.

In the present invention, the activated T cells can treat a cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

According to still yet another embodiment of the present invention, there may be provided an antigen-presenting cell (APC) loaded with a cancer-specific tumor antigen epitope provided in the present invention.

In the present invention, the antigen-presenting cells may include at least one of dendritic cell (DC), B cell, and macrophage, with dendritic cell being preferred.

In the present invention, the “dendritic cell” refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology and high expression levels of surface class I and class II MHC molecules, which are proteins that present antigenic peptides to T cells. DCs, other APCs, and T cells may be isolated or derived (such as differentiated) from a number of tissue sources, and conveniently from peripheral blood, such as peripheral blood mononuclear cells (PBMCs) derived from peripheral blood.

In the present invention, the antigen-presenting cell can induce differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, thereby treating a cancerous or neoplastic condition or preventing recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

According to still yet another embodiment of the present invention, there is provided a fusion protein, comprising: a cancer-specific tumor antigen epitope provided in the present invention; and a dendritic cell-specific antibody or a fragment thereof.

The fusion protein provided in the present invention enables the cancer-specific tumor antigen epitope provided in the present invention to be loaded on dendritic cells.

In the present invention, the dendritic cell-specific antibody may include, but is not limited to, antibodies specific for DCIR, MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, Clec9A, 33D1, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor, IL-2 receptor, ICAM-1, Fcγ receptor, LOX-1, or ASPGR, which is on dendritic cells.

The cancer-specific tumor antigen epitope in the fusion protein of the present invention may be conjugated to the dendritic cell-specific antibody or a fragment thereof. Here, the term “conjugate” refers to any material formed by joining two parts together. A representative conjugate according to the present invention includes those formed by joining an antigen together with an antibody and a TLR agonist. The term “conjugation” refers to a process of forming a conjugate and generally indicates physical coupling, for example, covalent bond, co-coordinate covalent bond, or second binding force, for example, Van der Waals binding force. The process of linking the antigen to the antibody may also be done via a non-covalent association such as a dockerin-cohesin association (as described in U.S. Patent Publication No. 20100135994, Banchereau et al. relevant portions incorporated herein by reference) or via a direct chemical linkage by forming a peptide or chemical bond.

According to another embodiment of the present invention, there is provided a method for producing an antigen-presenting cell (APC), in which the antigen-presenting cell is loaded with a cancer-specific tumor antigen epitope provided in the present invention.

In the present invention, the antigen-presenting cell may include one or more of dendritic cell, B cell, and macrophage, with dendritic cell being preferred.

In the present invention, the dendritic cells (such as immature dendritic cells) may be obtained from a variety of sources including autologous sources, that is, derived from a target individual. The dendritic cells may preferably be obtained from peripheral blood mononuclear cells (PBMCs) derived from peripheral blood, and more preferably be obtained by isolating monocytes from individual-derived PBMCs and contacting the monocytes with a plurality of cytokines. Here, the type of cytokine that induces differentiation of the monocytes into dendritic cells is not particularly limited, and may include, for example, one or more of GM-CSF and IL-4.

In the present invention, the “target individual” means an individual who has or is at high risk of developing cancer.

In the present invention, once antigen-presenting cells are prepared as described above, the antigen-presenting cells may be loaded with a cancer-specific tumor antigen epitope of the present invention. In general, immature dendritic cells capture an antigen through phagocytosis or receptor-mediated endocytosis, process the antigen through a series of intracellular processes and then cause an antigenic peptide to be loaded on MHC and presented to T lymphocytes. With the process of processing an antigen, the dendritic cells become more mature, which makes them lose receptors used for phagocytosis and endocytosis, exhibit increased expression of MHC class I, II, costimulatory molecules, and adhesion molecules, and express new chemokine receptors. This allows the dendritic cells to migrate to T lymphocyte-rich areas of the surrounding lymph nodes, and to present the antigen to T lymphocytes, thereby causing a T lymphocyte immune response.

In an example of the present invention, in order for the cancer-specific tumor antigen epitope to be loaded on the antigen-presenting cell, the antigen-presenting cell may be contacted with the cancer-specific tumor antigen epitope of the present invention, and preferably, a step of pulsing, with the cancer-specific tumor antigen epitope of the present invention, the antigen-presenting cells, for example, immature dendritic cells, or antigen-presenting cells (such as dendritic cells) contained in or derived (for example, differentiated) from PBMCs may be performed. As known in the art, pulsing refers to a process of mixing cells, such as dendritic cells, with a solution containing an antigenic peptide of the present invention, and then optionally removing the antigenic peptide from the mixture. In the present invention, when the immature dendritic cells are contacted with the cancer-specific tumor antigen epitope, treatment with toll-like receptor agonists may be performed to further induce maturation of a population of immature dendritic cells. Here, exemplary TLR agonists include, but are not limited to, polyIC, MALP, and R848.

In another example of the present invention, in order for the cancer-specific tumor antigen epitope to be loaded on the antigen-presenting cell, it is possible to perform nucleofection of the antigen-presenting cell with an expression vector, preferably a plasmid, into which a nucleic acid molecule encoding the cancer-specific tumor antigen epitope is inserted. Here, the nucleofection may be performed by any useful means in the art, including, for example, Amaxa® nucleofection system or InVitrogen® nucleofection system.

In yet another example of the present invention, in order for the cancer-specific tumor antigen epitope to be loaded on the antigen-presenting cell, such loading may be performed using a fusion protein that contains the cancer-specific tumor antigen epitope provided in the present invention; and a dendritic cell-specific antibody or a fragment thereof.

According to still yet another embodiment of the present invention, there is provided a T cell activated by an antigen-presenting cell provided in the present invention.

In the present invention, the T cells refer to a population of monoclonal (for example, encoding the same TCR) or polyclonal (for example, having clones encoding different TCRs) T cells that have T cell receptors recognizing a tumor antigen peptide, and may include one or more subtypes of T cells, including, but not limited to, one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells, with memory T cells being preferred.

In the present invention, the “memory T cells” are T cells that have previously encountered and responded to their specific antigen, or T cells that have differentiated from activated T cells. Although tumor-specific memory T cells make up a small portion of the total T cell amount, they play an important function in surveillance of tumor cells during a person's entire lifespan. In a case where tumor-specific memory T cells encounter tumor cells that express their specific tumor antigen, the memory T cells are immediately activated and clonally expanded. The activated and expanded T cells differentiate into effector T cells to kill tumor cells with high efficiency. Memory T cells are important for establishing and maintaining long-term tumor antigen-specific responses of T cells. In the present invention, activated T cells, preferably activated memory T cells, specifically recognize antigens on cancer cells, so that such T cells can treat a cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

According to still yet another embodiment of the present invention, there is provided a method for activating T cells using an antigen-presenting cell (APC) provided in the present invention.

In the present invention, for activation of the T cells, the T cells may be co-cultured with antigen-presenting cells loaded with a cancer-specific tumor antigen epitope of the present invention.

In the present invention, the T cells may be obtained from various sources including autologous sources, that is, derived from a target individual, may preferably be obtained from peripheral blood mononuclear cells (PBMCs) derived from peripheral blood, and may more preferably be obtained from non-adherent portions of the peripheral blood mononuclear cells. In an example of the present invention, the non-adherent portions of the PBMCs may be obtained by density gradient centrifugation of a peripheral blood sample, or may be obtained by performing culture with at least one cytokine (such as IL-2) in the presence or absence of an anti-CD3 antibody (such as OKT3).

In the present invention, the T cells refer to a population of monoclonal (for example, encoding the same TCR) or polyclonal (for example, having clones encoding different TCRs) T cells that have T cell receptors recognizing a tumor antigen peptide, and may include one or more subtypes of T cells, including, but not limited to, one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells, with memory T cells being preferred.

In addition, in the present invention, the T cells and the antigen-presenting cells may be derived from the same individual, such as an individual suffering from cancer (for example, low to medium grade cancer). However, the present invention is not limited thereto.

In the present invention, for activation of the T cells, the T cells may be co-cultured with antigen-presenting cells of the present invention for any one or more time periods of 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days, and preferably for 1 to 21 days, 1 to 14 days, 2 to 10 days, 2 to 5 days, 2 to 5 days, 3 days, 5 days, 7 days, 10 days, 14 days, 16 days, 18 days, or 21 days. However, the present invention is not limited thereto.

In the present invention, during the co-culture of the T cells with antigen-presenting cells of the present invention, one or more cytokines may be added to prime the T cells so that activation, maturation and/or proliferation of the T cells are promoted and the T cells subsequently differentiate into memory T cells. Exemplary cytokines that may be used at this stage include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), or combinations thereof, and the like.

In addition, in the present invention, during the co-culture of the T cells with antigen-presenting cells of the present invention, a fusion protein comprising a cytokine and an immunoglobulin heavy chain constant region may be added to prime the T cells so that activation, maturation and/or proliferation of the T cells are promoted and the T cells subsequently differentiate into memory T cells. Here, the cytokine may include, but is not limited to, interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), interleukin-18 (IL-18), and tumor necrosis factor (TNF), or granulocyte macrophage colony stimulating factor (GMCSF). The immunoglobulin heavy chain constant region may also be, but is not limited to, an immunoglobulin hinge region, and an immunoglobulin heavy chain constant region optionally selected from the group consisting of CH2 domain, CH3 domain, and CH4 domain, or combinations thereof. In addition, the immunoglobulin heavy chain constant region may be derived from immunoglobulins belonging to any of five immunoglobulin classes called in the art as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ), and may preferably be an immunoglobulin heavy chain constant region derived from the IgG class.

In addition, in the present invention, during the co-culture of the T cells with antigen-presenting cells of the present invention, a fusion protein that contains ligand binding to a cell surface protein which is highly expressed in memory T cells; and an immunoglobulin heavy chain constant region, may be added to prime the T cells so that activation, maturation and/or proliferation of the T cells are promoted and the T cells subsequently differentiate into memory T cells. Here, the cell surface protein which is highly expressed in memory T cells may be CD27, CXCR3, or CD62L. The ligand capable of binding to CD27 may be CD70; the ligand capable of binding to CXCR3 may be CXCR9 or CXCR10; and the ligand capable of binding to CD62L may be GlyCAM-1, CD34, MadCAM-1, or PSGL-1. However, the present invention is not limited thereto. In addition, the immunoglobulin heavy chain constant region may be derived from immunoglobulins belonging to any of five immunoglobulin classes called in the art as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ), and may preferably be an immunoglobulin heavy chain constant region derived from the IgG class.

According to still yet another embodiment of the present invention, there is provided an immunotherapeutic agent, comprising, as an active ingredient, an antigen-presenting cell loaded with a cancer-specific tumor antigen epitope provided in the present invention. The immunotherapeutic agent according to the present invention can increase immune responses or may selectively increase some of immune responses desired for treatment or prevention of a certain disease, for example, cancer.

According to still yet another embodiment of the present invention, there is provided an anticancer vaccine or a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, an antigen-presenting cell loaded with a cancer-specific tumor antigen epitope provided in the present invention; and/or an activated T cell.

The antigen-presenting cell provided in the present invention enables induction of differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, and the memory T cells thus activated can treat a cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

As used herein, the term “cancer” refers to or indicates a physiological condition characterized by cell growth in mammals which is not regulated in a typical manner. The cancer to be prevented, ameliorated, or treated in the present invention may be Epstein-Barr virus (EBV)-negative cancer, including, without limitation, any cancer species as long as it expresses a neoepitope represented by any one of SEQ ID NOs: 1 to 214 of the present invention. Thus, the type thereof is not particularly limited, and examples thereof may include, but are not limited to, colorectal cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, ovarian cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma, with gastric cancer being preferred.

In the present invention, the “prevention” may include, without limitation, any act of blocking symptoms of cancer, or suppressing or delaying the symptoms, using the pharmaceutical composition of the present invention.

In addition, in the present invention, the “treatment” may include, without limitation, any act of ameliorating or beneficially altering symptoms of cancer, using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized by being in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be characterized by being targeted to humans.

In the present invention, the pharmaceutical composition may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, and aqueous suspensions, preparations for external use, suppositories, and sterile injectable solutions, respectively, according to conventional methods, and used. However, the pharmaceutical composition is not limited thereto. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, a binder, a glidant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a flavor, and the like may be used for oral administration; a buffer, a preserving agent, a pain-relieving agent, a solubilizer, an isotonic agent, a stabilizer, and the like may be used in admixture for injections; and a base, an excipient, a lubricant, a preserving agent, and the like may be used for topical administration. The preparations of the pharmaceutical composition of the present invention may be prepared in various ways by being mixed with the pharmaceutically acceptable carrier as described above. For example, for oral administration, the pharmaceutical composition may be formulated in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, or the like. For injections, the pharmaceutical composition may be formulated in the form of unit dosage ampoules or multiple dosage forms. Alternatively, the pharmaceutical composition may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, or the like.

Meanwhile, as examples of carriers, diluents, or excipients suitable for making preparations, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, or the like may be used. In addition, a filler, an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like may further be included.

The route of administration of the pharmaceutical composition of the present invention includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal route. Oral or parenteral administration is preferred.

As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrabursal, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention may vary depending on a variety of factors, including activity of a certain compound used, the patient's age, body weight, general health status, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and severity of a certain disease to be prevented or treated. A dose of the pharmaceutical composition may vary depending on the patient's condition, body weight, severity of disease, drug form, route of administration, and duration, and may be appropriately selected by those skilled in the art. The pharmaceutical composition may be administered in an amount of 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg, per day. Administration may be made once a day or several times a day. The dose is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

According to still yet another embodiment of the present invention, there is provided a method for preventing or treating cancer, comprising a step of administering, to a target individual, an antigen-presenting cell loaded with a cancer-specific tumor antigen epitope provided in the present invention; and/or an activated T cell.

Dose, schedule, and route of administration of the antigen-presenting cell loaded with a cancer-specific tumor antigen epitope provided in the present invention or the activated T cell may be determined depending on the size and condition of an individual, and in accordance with standard pharmaceutical practice. Exemplary routes of administration include intravenous, intraarterial, intraperitoneal, intrapulmonary, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, or transdermal route.

A dose of cells administered to an individual may vary depending, for example, on the particular type of cells being administered, the route of administration, and the particular type and stage of cancer being treated. The amount should be sufficient to produce a desirable response, such as a therapeutic response against cancer, but without severe toxicity or adverse events. In some embodiments, the amount of activated T cells or antigen-presenting cells (such as dendritic cells) to be administered is a therapeutically effective amount. In some embodiments, the amount of cells (such as dendritic cells loaded with a cancer-specific tumor antigen epitope or activated T cells) is an amount sufficient to decrease the size of a tumor, decrease the number of cancer cells, or decrease the growth rate of a tumor by any one of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, as compared with the corresponding tumor size, number of cancer cells, or tumor growth rate in the same individual prior to treatment or as compared with the corresponding activity in other individuals having not received the treatment. The magnitude of effects may be measured using standard methods, such as in vitro assays with purified enzymes, cell-based assays, animal models, or experiments using humans.

In an embodiment of the present invention, the antigen-presenting cells (such as dendritic cells) loaded with a cancer-specific tumor antigen epitope of the present invention may be administrated at a dose of any of 1×10⁵ to 5×10⁵, 5×10⁵ to 1×10⁶, 1×10⁶ to 2×10⁶, 2×10⁶ to 3×10⁶, 3×10⁶ to 4×10⁶, 4×10⁶ to 5×10⁶, 5×10⁶ to 6×10⁶, 6×10⁶ to 7×10⁶, 7×10⁶ to 8×10⁶, 8×10⁶ to 1×10⁸, 1×10⁶ to 3×10⁶, 3×10⁶ to 5×10⁶, 5×10⁶ to 7×10⁶, 2×10⁶ to 4×10⁶, 1×10⁶ to 5×10⁶, or 5×10⁶ to 1×10⁷ cells/individual. However, the present invention is not limited thereto.

In another embodiment of the present invention, the antigen-presenting cells (e.g., dendritic cells) loaded with a cancer-specific tumor antigen epitope of the present invention may be administrated at a dose of any of 1×10⁴ to 5×10⁴, 5×10⁴ to 1×10⁵, 1×10⁵ to 2×10⁵, 2×10⁵ to 4×10⁵, 4×10⁵ to 6×10⁵, 6×10⁵ to 8×10⁵, 8×10⁵ to 1×10⁶, 1×10⁶ to 2×10⁶, 2×10⁶ to 1×10⁷, 1×10⁴ to 1×10⁵, 1×10⁵ to 1×10⁶, 1×10⁶ to 1×10, 1×10⁴ to 1×10⁶, or 1×10⁵ to 1×10⁷ cells/kg. However, the present invention is not limited thereto.

In addition, in an embodiment of the present invention, the activated T cells of the present invention may be administrated at a dose of any of 1×10⁸ to 5×10⁸, 5×10⁸ to 9×10⁸, 9×10⁸ to 1×10⁹, 1×10⁹ to 2×10⁹, 2×10⁹ to 3×10⁹, 3×10⁹ to 4×10⁹, 4×10⁹ to 5×10⁹, 5×10⁹ to 6×10⁹, 6×10⁹ to 1×10¹⁰, 1×10⁹ to 3×10⁹, 3×10⁹ to 5×10⁹, 5×10⁹ to 7×10⁹, 7×10⁹ to 1×10¹⁰, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 3×10⁹ to 7×10⁹, 1×10¹⁰ to 1.5×10¹⁰, 1×10¹⁰ to 2×10¹⁰, or 1×10⁹ to 1×10¹⁰ cells/individual. However, the present invention is not limited thereto.

In another embodiment of the present invention, the activated T cells of the present invention may be administrated at a dose of any of 1×10⁷ to 1×10⁸, 1×10⁸ to 2×10⁸, 2×10⁸ to 4×10⁸, 4×10⁸ to 6×10⁸, 6×10⁸ to 8×10⁸, 8×10⁸ to 1×10⁹, 1×10⁹ to 2×10⁹, 2×10⁹ to 4×10⁹, 4×10⁹ to 1×10¹⁰, 2×10⁸ to 6×10⁸, 6×10⁸ to 1×10⁹, 1×10⁸ to 2×10⁸, 2×10⁸ to 2×10⁹, 1×10⁷ to 1×10⁸, 1×10⁸ to 1×10⁹, 1×10⁹ to 1×10¹⁰, or 1×10⁷ to 1×10⁹ cells/kg. However, the present invention is not limited thereto.

In the present invention, a stabilizer or excipient such as human albumin may be used together with administration of the antigen-presenting cells (such as dendritic cells) loaded with a cancer-specific tumor antigen epitope and/or the activated T cells.

In the present invention, dose and dosing schedule of the antigen-presenting cells (such as dendritic cells) loaded with a cancer-specific tumor antigen epitope and/or the activated T cells may be adjusted over the course of treatment based on the judgment of the administering physician. In some embodiments, the activated T cells may be administered at any time point of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 1 month after the antigen-presenting cells loaded with a tumor antigen peptide are administered, or may be administered simultaneously with the antigen-presenting cells. However, the present invention is not limited thereto.

In the present invention, administration of the antigen-presenting cells (such as dendritic cells) loaded with a cancer-specific tumor antigen epitope and/or the activated T cell may be done alone or in combination with other therapies, such as surgery, radiation therapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, hormone therapy, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, chemotherapy, or the like. Additionally, a person having a greater risk of developing a proliferative disease may receive treatments to inhibit and/or delay development of the disease.

ADVANTAGEOUS EFFECTS OF INVENTION

The antigen-presenting cell, that is, dendritic cell, loaded with a cancer-specific tumor antigen epitope provided in the present invention enables rapid and effective induction of differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, and the memory T cells thus activated can treat cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

In the conventional adoptive T cell therapies, it takes a long time of 3 to 6 months to produce a large number of T cells for treatment of cancer patients, which poses a big problem in the cell production process in immune cell therapy. However, according to the present invention, 10⁹ autologous memory T cells, which should be used for patient treatment, can be produced within three weeks, and cost reduction and minimized infection risk factors to external contaminants can be achieved. Accordingly, according to the present invention, there is provided a technique that can be applied to terminal cancer patients because such a technique makes rapid therapeutic approaches available for a larger number of solid cancer patients.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates results obtained by identifying, through IFN-γ ELISPOT, a proportion of cells that secrete IFN-γ, in T cells stimulated with dendritic cells loaded with a neoepitope (10-mer) of EBV-negative gastric cancer antigen, and unstimulated control cells, to identify antigen specificity of EBV-negative gastric cancer-specific autologous memory T cells (HLA-A2402) produced according to an embodiment of the present invention.

FIG. 2 illustrates results obtained by identifying, through IFN-γ ELISPOT, a proportion of cells that secrete IFN-γ, in T cells stimulated with dendritic cells loaded with a neoepitope (10-mer) of EBV-negative gastric cancer antigen, and unstimulated control cells, to identify antigen specificity of EBV-negative gastric cancer-specific autologous memory T cells (HLA-A2402) produced according to an embodiment of the present invention.

FIG. 3 illustrates results obtained by identifying, through IFN-γ ELISPOT, a proportion of cells that secrete IFN-γ, in T cells stimulated with dendritic cells loaded with a neoepitope (10-mer) of EBV-negative gastric cancer antigen, and unstimulated control cells, to identify antigen specificity of EBV-negative gastric cancer-specific autologous memory T cells (HLA-A0201) produced according to an embodiment of the present invention.

FIG. 4 illustrates results obtained by identifying, through IFN-γ ELISPOT, a proportion of cells that secrete IFN-γ, in T cells stimulated with dendritic cells loaded with a neoepitope (10-mer) of EBV-negative gastric cancer antigen, and unstimulated control cells, to identify antigen specificity of EBV-negative gastric cancer-specific autologous memory T cells (HLA-A0201) produced according to an embodiment of the present invention.

DETAILED DESCRIPTION OF INVENTION

According to an embodiment of the present invention, there is provided an Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope, represented by any one of SEQ ID NOs: 1 to 214.

According to another embodiment of the present invention, there is provided an antigen-presenting cell (APC) loaded with a cancer-specific tumor antigen neoepitope provided in the present invention.

According to yet another embodiment of the present invention, there is provided a T cell activated by an antigen-presenting cell provided in the present invention.

According to still yet another embodiment of the present invention, there is provided an anticancer vaccine or a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, an antigen-presenting cell loaded with a cancer-specific tumor antigen epitope provided in the present invention; and/or an activated T cell.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited by these examples.

EXAMPLES Example 1 Production Method of Autologous Memory T Cells Specific to EBV-Negative Gastric Cancer Cells and Clinical Application Thereof

1. Selection of EBV-Negative Gastric Cancer Cell Antigen Neoepitopes

Algorithms for predicting the most important sequence with accumulation of genetic mutations in gastric cancer cells and for predicting epitopes of this sequence which bind to HLA of T cells were developed using Neopepsee. Here, using the neoepitope prediction algorithms, peptide sequences were identified which are expected to have high binding affinity with HLA types (HLA-A2402, HLA-A0201) that Koreans express the most. To this end, missense mutations expressed into mRNAs were predicted through whole-exome sequencing and RNAseq data analysis of all EBV-negative gastric cancer patients currently present in TCGA. For binding affinity between each HLA type and the identified neoepitope, Neopepsee final scores were calculated considering both IC₅₀ values (nM) obtained from NetMHC and rank-based predictive values of MHC-peptide binding obtained from NetCTLpan, and further considering all of protein cleavage, hydrophobicity of amino acids in TCR contact residues, polarity and charged values of amino acids, and molecular size and peptide entropy. In this way, possibility of cancer-specific neoantigens was finally predicted, so that neoepitopes were identified.

Tables 1 to 6 below show sequences that are expected to be neoepitopes, corresponding genes expressing the sequences, and normal sequences, obtained by analysis of RNAseq data of patients expressing HLA-A2402 and HLA-A0201 which are known to be most expressed in Koreans, which provides prediction of binding affinity between each of the present sequences and HLA, and thus provides prediction of types of neoepitopes that can be used when producing actual cell therapeutic agents.

TABLE 1 Neoepitope (8-mer) with high binding affinity for HLA-A2402 expr Score transcript gene chr pos ref alt (RPKM) WT_AA MT_AA MTpep MT_ic50 level hg19_ MYO1D chr17 31087632 C T 0 FAKAI FAKAI SEQ ID IYEH 379 medium knownGene_ YERLF YEHLF NO: 1 LFC uc002hhp.1, CWIV CWIV W hg19_ T T knownGene_ uc002hho.1, hg19_ knownGene_ uc010wcb.2

TABLE 2 Neoepitopes (9-mer) with high binding affinity for HLA-A2402 expr Score transcript gene chr pos ref alt (RPKM) WT_AA MT_AA MTpep MT_ic50 level hg19_ STRIP2 chr7 1.29E+08 C T 0 MSAIYQ MSAIYQ SEQ ID IYQ 35 high knownGene_ KVRHR KVCHR NO: 2 KV uc011koz.2, MNDD MNDD CH hg19_ WA WA RM knownGene_ uc003vow.3, hg19_ knownGene_ uc011koy.2 hg19_ SLC32A1 chr20 37357184 G A 0 LWHQV LWHQV SEQ ID LW 70 high knownGene_ FFDVAI FFDIAIF NO: 3 HQ uc002xjc.3 FVIGGI VIGGI VFF DI hg19_ SIRT2 chr19 39380364 C T 0 KKHPEP KKHPEP SEQ ID KH 132 high knownGene_ FFALAK FFTLAK NO: 4 PEP uc010egi.2, ELYPG ELYPG FFT hg19_ L knownGene_ uc002ojt.2, hg19_ knownGene_ uc002ojv.2, hg19_ knownGene_ uc002ojs.2, hg19_ knownGene_ uc002oju.2, hg19_ knownGene_ uc010egh.2 hg19_ ERBB2 chr17 37881332 G A 0 SYLEDV SYLEDV SEQ ID SY 54 high knownGene_ RLVHR RLIHRD NO: 5 LE uc002hso.3, DLAAR LAARN DV hg19_ N RLI knownGene_ uc010cwa.3, hg19_ knownGene_ uc002hsp.3, hg19_ knownGene_ uc010cwb.3, hg19_ knownGene_ uc002hsm.3, hg19_ knownGene_ uc010wek.2 hg19_ CASC3 chr17 38325584 C T 0 LYPNTQ LYPNTQ SEQ ID LYP 184 high knownGene_ APSQV APLQV NO: 6 NT uc002hue.3, YGGVT YGGVT QA hg19_ Y Y PL knownGene_ uc010cwt.1 hg19_ TM2D3 chr15 1.02E+08 G A 0 SFGGLG SFGGLG SEQ ID IW 394 high knownGene_ IWTLID IWMLID NO: 7 MLI uc002bxi.3, VLLIG VLLIG DV hg19_ LL knownGene_ c002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ PLD4 chr14 1.05E+08 G A 0 QNFSSH QNFSSH SEQ ID NFS 83 medium knownGene_ FNRFQP FNHFQP NO: 8 SHF uc001ypu.1, FHGLF FHGLF NH hg19_ F knownGene_ uc010tyl.1 hg19_ PLD4 chr14 1.05E+08 G A 0 QNFSSH QNFSSH SEQ ID HF 89 high knownGene_ FNRFQP FNHFQP NO: 9 QPF uc001ypu.1, FHGLF FHGLF HG hg19_ LF knownGene_ uc010tyl.1 hg19_ PCDHB6 chr5 1.41E+08 C T 0 INAITG INAITG SEQ ID IW 36 high knownGene_ EIRLRK EIWLRK NO: 10 LR uc003lir.3 ALDFE ALDFE KA LD F hg19_ CST4 chr20 23669411 G A 0 ATEDEY ATEDEY SEQ ID YY 159 high knownGene_ YRRPLQ YRCPLQ NO: 11 RC uc002wto.1 VLRAR VLRAR PL QV L hg19_ C6 chr5 41149516 T G 0 DYFTSP DYFTSP SEQ ID YF 127 high knownGene_ ACKFL ACTFLA NO: 12 TSP uc003jml.2, AEKCL EKCLN AC hg19_ N TF knownGene_ uc003jmk.3 hg19_ AFAP1L1 chr5 1.49E+08 C T 0 CRICAF CRICAF SEQ ID AF 325 medium knownGene_ LLRKK LLWKK NO: 13 LL uc003lqh.3, RFGQW RFGQW WK hg19_ A A KR knownGene_ F uc010jgy.3, hg19_ knownGene_ uc003lqg.4 hg19_ ACVR1B chr12 52387827 G A 0 CWYAN CWYAN SEQ ID WY 219 medium knownGene_ GAARLT GAAHL NO: 14 AN uc021qya.1, ALRIKK TALRIK GA hg19_ K AH knownGene_ L uc001rzn.3, hg19_ knownGene_ uc010snn.2 hg19_ ZNF83 chr19 53116566 A G 0 FSQNSY FSQNSY SEQ ID SY 10 high knownGene_ LAYHW LAHHW NO: 15 LA uc010epz.3, RIHTGE RIHTGE HH hg19_ WR knownGene_ I uc021uyx.1, hg19_ knownGene_ uc010epx.3, hg19_ knownGene_ uc010epy.3, hg19_ knownGene_ uc010eps.3, hg19_ knownGene_ uc010epv.3, hg19_ knownGene_ uc010epw.3, hg19_ knownGene_ uc010eqb.2, hg19_ knownGene_ uc002pzu.4, hg19_ knownGene_ uc002pzv.4, hg19_ knownGene_ uc031rmq.1, hg19_ knownGene_ uc031rmp.1, hg19_ knownGene_ uc031rmm.1, hg19_ knownGene_ uc031rmn.1, hg19_ knownGene_ uc031rml.1, hg19_ knownGene_ uc031rmo.1, hg19_ knownGene_ uc010epu.3, hg19_ knownGene_ uc010ept.3 hg19_ SLC9A1 chr1 27436202 C T 0 VGIVDI VGIVDI SEQ ID IFL 59 high knownGene_ FLGFLS FLSFLS NO: 16 SFL bnm.4, FFVVA FFVVA SFF hg19_ knownGene_ uc001bnn.3 hg19_ SLC6A15 chr12 85279797 G A 0 MVIGIP MVIGIP SEQ ID MV 216 high knownGene_ LFFLEL FFFLEL NO: 17 IGI sul.3 SVG SVG PFF F hg19_ SLC6A15 chr12 85279797 G A 0 LLMVIG LLMVIG SEQ ID MV 216 high knownGene_ IPLFFLE IPFFFLE NO: 18 IGI uc001szy.4, LSVG LSVG PFF hg19_ F knownGene_ uc001szv.4 hg19_ SARDH chr9 1.37E+08 G A 0 MSLGK MSLGK SEQ ID VY 161 high knownGene_ AYGVES VYGVE NO: 19 GV uc011mdo.2 HVL SHVL ES HV L hg19_ SARDH chr9 1.37E+08 G A 0 KRLMS KRLMS SEQ ID VY 161 high knownGene_ LGKAY LGKVY NO: 20 GV uc004cep.4, GVESH GVESH ES hg19_ VL VL HV knownGene_ L uc004ceo.3, hg19_ knownGene_ uc011mdn.2 hg19_ OPRK1 chr8 54142147 C T 0 LVVVAV LVVVAV SEQ ID VF 297 high knownGene_ FVVCW FVICWT NO: 21 VIC uc003xrh.12, TPIHIF PIHIF WT hg19_ PI knownGene_ uc022aup.1, hg19_ knownGene_ uc010lyc.1, hg19_ knownGene_ uc003xri.1 hg19_ MYO1D chr17 31087632 C T 0 AFAKAI AFAKAI SEQ ID IYE 195 high knownGene_ YERLFC YEHLFC NO: 22 HL uc002hhp.1 WIVTR WIVTR FC hg19_ WI knownGene_ uc002hho.1, hg19_ knownGene_ uc010wcb.2 hg19_ KLK8 chr19 51503767 G A 0 QPHSQP QPHSQP SEQ ID SQP 132 high knownGene_ WQAAL WQVAL NO: 23 WQ uc002puu.1, FQGQQ FQGQQ VA hg19_ L L LF knownGene_ uc002puq.1, hg19_ knownGene_ uc002pur.1 hg19_ IRAK2 chr3 10264468 C T 0 AYLPED AYLPED SEQ ID AY 364 high knownGene_ FIRVGQ FIWVG NO: 24 LPE uc003bve.1 LTKRV QLTKRV DFI W hg19_ HS3ST2 chr16 22926539 G A 0 NAIRIG NAIRIG SEQ ID MY 23 high knownGene_ MYVLH MYMLH NO: 25 ML uc002dli.3 LESWL LESWL HL Q Q ES W hg19_ HOXA7 chr7 27194754 G A 0 EFHFNR EFHFNR SEQ ID RY 73 medium knownGene_ YLTRRR YLMRR NO: 26 LM uc003sys.3 RIEIA RRIEIA RR RRI hg19_ DOCK5 chr8 25158099 T C 0 QSTFISE QSTFISE SEQ ID TFI 63 high knownGene_ NYLIRW NHLIRW NO: 27 SE uc003xeg.3, GSNG GSNG NH hg19_ LI knownGene_ uc003xef.3 hg19_ CNR1 chr6 88853864 G A 0 GKMNK GKMNK SEQ ID KLI 208 high knownGene_ LIKTVF LIKMVF NO: 28 KM uc011dzr.2, AFCSM AFCSM VF hg19_ L L AF knownGene_ uc011dzt.2, hg19_ knownGene_ uc010kbz.3, hg19_ knownGene_ uc010kca.3, hg19_ knownGene_ uc003pmq.4, hg19_ knownGene_ uc011dzs.2, hg19_ knownGene_ uc021zco.1 hg19_ CARD11 chr7 2956965 G A 0 TSDPRV TSDPRV SEQ ID CLS 35 high knownGene_ SPRLSR SPCLSR NO: 29 RA uc003smv.3 ASFLF ASFLF SFL F hg19_ ABCB5 chr7 20782555 G A 0 EVSFFY EVSFFY SEQ ID FYP 51 medium knownGene_ PCRPDV PCHPDV NO: 30 CH uc003suw.4, FILRG FILRG PD hg19_ VF knownGene_ uc010kuh.3

TABLE 3 Neoepitopes (10-mer) with high binding affinity for HLA-A2402 expr Score transcript gene chr pos ref alt (RPKM) WT_AA MT_AA MTpep MT_ic50 level hg19_ RAB40B chr17 80616484 C T 0 AQAYA AQAYA SEQ AYA  19 high knownGene_ ERLGVT ERLGM ID ERL uc002kft.3 FFEVSP TFFEVS NO: GMT LC PLC 31 F hg19_ CRB1 chr1 1.97E+08 T G 0 TSNGVA TSNGVA SEQ VYN  38 high knownGene_ LLNFYN LLNVY ID MPS uc010ppd.2, MPSTPS NMPSTP NO: TPSF hg19_ F SF 32 knownGene_ uc009wza.3, hg19_ knownGene_ uc001gtz.3, hg19_ knownGene_ uc010ppb.2, hg19_ knownGene_ uc010poz.2, hg19_ knownGene_ uc001gub.1 hg19_ WDR91 chr7 1.35E+08 C T 0 LRDYW LRDYW SEQ YWS  40 high knownGene_ SYLERR SYLEHR ID YLE uc003vsp.2 LFSRLE LFSRLE NO: HRL DI DI 33 F hg19_ TM2D3 chr15 1.02E+08 G A 0 FSFGGL FSFGGL SEQ IWM  47 high knownGene_ GIWTLI GIWMLI ID LIDV uc002bxi.3, DVLLIG DVLLIG NO: LLI hg19_ V V 34 knownGene_ uc002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ C6 chr5 41149516 T G 0 NDYFTS NDYFTS SEQ DYF  60 medium knownGene_ PACKFL PACTFL ID TSPA uc003jml.2, AEKCL AEKCL NO: CTF hg19_ NN NN 35 knownGene_ uc003jmk.3 hg19_ ABCA5 chr17 67257696 G A 0 FWSFIY FWSFIY SEQ IYSV  64 high knownGene_ SVAALA SVAVLA ID AVL uc002jig.2, CIAITEI CIAITEI NO: ACI hg19_ 36 knownGene_ uc002jid.2, hg19_ knownGene_ uc002jib.2, hg19_ knownGene_ uc002jic2, hg19_ knownGene_ uc002jif.2 hg19_ SF3A1 chr22 30730630 C T 0 LAYYN LAYYN SEQ YYN 80 medium knownGene_ MANGA MANGT ID MAN uc003ahl.3, VIHLAL VIHLAL NO: GTVI hg19_ KER KER 37 knownGene_ uc021wnt.1 hg19_ CSPG4 chr15 75974714 G A 0 PQLLLY PQLLLY SEQ VWG 110 high knownGene_ RVVRGP RVVWG ID PQL uc002baw.3 QLGRLF PQLGRL NO: GRL H FH 38 F hg19_ ABCB5 chr7 20782555 G A 0 REVSFF REVSFF SEQ FYPC 111 high knownGene_ YPCRPD YPCHPD ID HPD uc003suw.4, VFILRG VFILRG NO: VFI knownGene_ L L 39 uc010kuh.3 hg19_ KCNMB2 chr3 1.79E+08 T G 0 CSYIPK CSYIPK SEQ SYIP 133 medium knownGene_ CGKNF CGKKF ID KCG uc003fje.3, EESMSL EESMSL NO: KKF hg19_ VN VN 40 knownGene_ uc031scj.1, hg19_ knownGene_ uc003fjd.3, hg19_ knownGene_ uc003fjf.3 hg19_ ARMC2 chr6 1.09E+08 G T 0 IKKLVD IKKLVD SEQ RYL 159 high knownGene_ CLRDL CLRYLG ID GPT uc011eao.2, GPTDW PTDWQ NO: DWQ hg19_ QLA LA 41 L knownGene_ uc003pss.4 hg19_ CDH1 chr16 68844172 G T 0 PMEILIT PMEILIT SEQ TYQ 187 high knownGene_ VTDQN VTYQN ID NDN uc002ewg.1, DNKPEF DNKPEF NO: KPEF hg19_ T T 42 knownGene_ uc010cfg.1 hg19_ HS3ST2 chr16 22926539 G A 0 WNAIRI WNAIRI SEQ MYM 203 high knownGene_ GMYVL GMYML ID LHLE uc002dli.3 HLESW HLESW NO: SWL LQY LQY 43 hg19_ SLC32A1 chr20 37357184 G A 0 LLWHQ LLWHQ SEQ VFF 269 high knownGene_ VFFDVA VFFDIAI ID DIAI uc002xjc.3 IFVIGGI FVIGGI NO: FVI C C 44 hg19_ ITGB1 chr10 33209310 G T 0 QLIIDA QLIIDA SEQ AYN 274 medium knownGene_ YNSLSS YNSISS ID SISS uc001iwr.4, EVILEN EVILEN NO: EVI hg19_ G G 45 knownGene_ uc001iwt.4, hg19_ knownGene_ uc001iws.4 hg19_ RTFDC1 chr20 55059189 G A 0 HRFCFL HRFCFL SEQ CFLR 316 high knownGene_ RCCGC RCCSCV ID CCS uc010zzf.1, VFSERA FSERAL NO: CVF hg19_ LK K 46 knownGene_ uc002xxt.2, hg19_ knownGene_ uc002xxu.2 hg19_ CST4 chr20 23669411 G A 0 KATEDE KATEDE SEQ EYY 370 medium knownGene_ YYRRPL YYRCPL ID RCPL uc002wto.1 QVLRA QVLRA NO: QVL RE RE 47 hg19_ SLC6A15 chr12 85279797 G A 0 ILLMVI ILLMVI SEQ LMV 404 high knownGene_ GIPLFFL GIPFFFL ID IGIPF uc001szy.4, ELSVGQ ELSVGQ NO: FF hg19_ 48 knownGene_ uc001szv.4 hg19_ ABCB5 chr7 20782555 G A 0 REVSFF REVSFF SEQ FFYP 436 medium knownGene_ YPCRPD YPCHPD ID CHP uc003suw.4, VFILRG VFILRG NO: DVF hg19_ L L 49 knownGene_ uc010kuh.3

TABLE 4 Neoepitopes (8-mer) with high binding affinity for HLA-A0201 expr Score transcript gene chr pos ref alt (RPKM) WT_AA MT_AA MTpep MT_ic50 level hg19_ TM2D3 chr15 1.02E+08 G A 0 FGGL FGGL SEQ MLI  52 high knownGene_ GIWTL GIWM ID DV uc002bxi.3, IDVLL LIDVL NO: LLI hg19_ I LI 50 knownGene_ uc002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ FASN chr17 80049217 G A 0 LSML LSML SEQ ML  70 high knownGene_ NDIAA NDIVA ID NDI uc002kdu.3 VPATA VPATA NO: VA M M 51 V hg19_ SARDH chr9 1.37E+08 G A 0 MSLG MSLG SEQ SL 105 high knownGene_ KAYG KVYG ID GK uc011mdo.2 VESH VESH NO: VY V V 52 GV hg19_ SARDH chr9 1.37E+08 G A 0 RLMS RLMS SEQ SL 105 high knownGene_ LGKA LGKV ID GK uc004cep.4, YGVE YGVE NO: VY hg19_ SHV SHV 53 GV knownGene_ uc004ceo.3, hg19_ knownGene_ uc011mdn.2 hg19_ SIGLEC5 chr19 52131128 G A 0 FTCRA FTCRA SEQ HL 219 high knownGene_ QHPL QHLL ID LG uc002pxe.4 GFLQI GFLQI NO: FL F F 54 QI hg19_ NETO1 chr18 70451000 G A 0 VAND VAND SEQ ML 247 high knownGene_ VMLR VMLC ID CT uc002lkw.3, TGLG TGLG NO: GL hg19_ VIR VIR 55 GV knownGene_ uc002lky.2 hg19_ TM2D3 chr15 1.02E+08 G A 0 FGGL FGGL SEQ GL 329 high knownGene_ GIWTL GIWM ID GI uc002bxi.3, IDVLL LIDVL NO: WM hg19_ I LI 56 LI knownGene_ uc002bxh.3, hg19_ knownGene_ uc002bxj.3

TABLE 5 Neoepitopes (9-mer) with high binding affinity for HLA-A0201 expr Score transcript gene chr pos ref alt (RPKM) WT_AA MT_AA MTpep MT_ic50 level hg19_ SLC9A1 chr1  27436202 C T 0 VGIVD VGIVD SEQ FLS 2 high knownGene_ IFLGF IFLSFL ID FLS uc001bnm.4, LSFFV SFFVV NO: FFV hg19_ VA A 57 knownGene_ uc001bnn.3 hg19_ IRAK2 chr3  10264468 C T 0 AYLPE AYLPE SEQ YL 3 high knownGene_ DFIRV DFIW ID PE uc003bve.1 GQLT VGQL NO: DFI KRV TKRV 58 WV hg19_ FASN chr17  80049217 G A 0 FLSML FLSML SEQ SM 6 high knownGene_ NDIAA NDIVA ID LN uc002kdu.3 VPATA VPATA NO: DIV MP MP 59 AV hg19_ CLSTN1 chr1   9795564 G A 0 YLNS YLNS SEQ YL 7 high knownGene_ RQFPT RQFP ID NS uc001aqh.3, PGIRR MPGIR NO: RQ hg19_ LKI RLKI 60 FP knownGene_ M uc001aqi.3, hg19_ knownGene_ uc010oag.2 hg19_ STK36 chr2 219558685 G A 0 YFLSL YFLSL SEQ FLS 8 high knownGene_ LVFRL LVFQL ID LL uc002viv.3, QNLP QNLP NO: VF hg19_ CGM CGM 61 QL knownGene_ uc002viu.3 hg19_ HS3ST2 chr16  22926539 G A 0 NAIRI NAIRI SEQ YM 8 high knownGene_ GMYV GMYM ID LH uc002dli.3 LHLES LHLES NO: LES WLQ WLQ 62 WL hg19_ ARMC2 chr6 109286202 G T 0 KKLV KKLV SEQ KL 8 high knownGene_ DCLR DCLR ID VD uc011eao.2, DLGPT YLGPT NO: CL hg19_ DWQL DWQL 63 RY knownGene_ L uc003pss.4 6hg19_ RTN2 chr19  45997462 G A 0 VRGQ VRGQ SEQ SM 9 high knownGene_ CLDST CLDS ID DQ uc002pcc.4, DQLEF MDQL NO: LEF hg19_ TVE EFTVE 64 TV knownGene_ uc002pcb.4 hg19_ RAB40B chr17  80616484 C T 0 QAYA QAYA SEQ RL 13 high knownGene_ ERLG ERLG ID GM uc002kft.3 VTFFE MTFFE NO: TFF VSPL VSPL 65 EV hg19_ ARMC2 chr6 109286202 G T 0 KKLV KKLV SEQ YL 15 high knownGene_ DCLR DCLR ID GP uc011eao.2, DLGPT YLGPT NO: TD hg19_ DWQL DWQL 66 WQ knownGene_ L uc003pss.4 hg19_ ABCA10 chr17  67178331 G A 0 ALMG ALMG SEQ AL 16 high knownGene_ IFNFT IFNFM ID MG uc010dfa.1, ELIQM  ELIQM NO: IFN hg19_ EST EST 67 FM knownGene_ uc010dfb.1 hg19_ CYP4X1 chr1  47512210 C T 0 TCRLI TCRLI SEQ RLI 16 high knownGene_ PAVPSI PAVLS ID PAV uc001cqs.3, SRDLS ISRDL NO: LSI hg19_ K SK 68 knownGene_ uc001cqr.3, hg19_ knownGene_ uc001cqt.3 hg19_ SLC22A16 chr6 110746270 C T 0 PQLFV PQLFV SEQ TLL 20 high knownGene_ GTMA GTMT ID SG uc003pue.3, LLSGV LLSGV NO: VL hg19_ LTL LTL 69 TL knownGene_ uc003puf.3 hg19_ FASN chr17  80049217 G A 0 FLSML FLSML SEQ FLS 20 high knownGene_ NDIAA NDIVA ID ML uc002kdu.3 VPATA VPATA NO: NDI MP MP 70 V hg19_ NEDD9 chr6  11185718 C T 0 ISLLN ISLLN SEQ TLF 21 high knownGene_ AIDAL AIDTL ID SC uc010joz.2, FSCVS FSCVS NO: VSS hg19_ SA SA 71 A knownGene_ uc031sms.1, hg19_ knownGene_ uc003mzv.2, hg19_ knownGene_ uc003mzw.3 hg19_ NETO1 chr18  70451000 G A 0 TVAN TVAN SEQ VM 26 high knownGene_ DVML DVML ID LC uc002lkw.3, RTGL CTGL NO: TG hg19_ GVIR GVIR 72 LG knownGene_ M M V uc002lky.2 hg19_ KIF15 chr3  44828026 C T 0 KKGV KKGV SEQ FV 29 high knownGene_ FVVG FVVG ID VG uc010hiq.3, AVEQ VVEQ NO: VV hg19_ VVTS VVTS 73 EQ knownGene_ A A V uc003cnx.4 hg19_ FOXF1 chr16  86545019 G A 0 ASAA ASAA SEQ AL 29 high knownGene_ LNSG LNSGT ID NS uc002fjl.3 ASYIK SYIKQ NO: GT QQPL QPL 74 SYI hg19_ NEDD9 chr6  11185718 C T 0 ISLLN ISLLN SEQ SLL 30 high knownGene_ AIDAL AIDTL ID NAI uc010joz.2, FSCVS FSCVS NO: DT hg19_ SA SA 75 L knownGene_ uc031sms1, hg19_ knownGene_ uc003mzv.2, hg19_ knownGene_ uc003mzw.3 hg19_ EXO1 chr1 242035442 T C 0 KSLSF KSLSF SEQ SLS 32 high knownGene_ SEVFV SEVSV ID FSE uc001hzh.3, PDLVN PDLVN NO: VS hg19_ GP GP 76 V knownGene_ uc021plk.1, hg19_ knownGene_ uc009xgq.3, hg19_ knownGene_ uc021plj.1 hg19_ GNPDA1 chr5 141384531 G A 0 TKVPT TKVPT SEQ AL 33 high knownGene_ MALT MALM ID MV uc003lmh4, VGVG VGVG NO: GV hg19_ TVMD TVMD 77 GT knownGene_ V uc010jgh.3, hg19_ knownGene_ uc003lmf.4, hg19_ knownGene_ uc003lmg.4 hg19_ ABCA5 chr17  67257396 G A 0 WSFIY WSFIY SEQ FIY 38 high knownGene_ SVAAL SVAAVL ID SV uc002jig.2, ACIAI ACIAI NO: knownGene_ TE TE 78 LA uc002jid.2, hg19_ knownGene_ uc002jib.2, hg19_ knownGene_ uc002jic.2, hg19_ knownGene_ uc002jif.2 hg19_ TM2D3 chr15 102182749 G A 0 SFGGL SFGGL SEQ WM 41 high knownGene_ GIWTL GIWM ID LID uc002bxi.3, IDVLL LIDVL NO: VL hg19_ IG LIG 79 LI knownGene_ uc002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ SOX6 chr11  16077437 G A 0 HKQIE HKQIE SEQ KQI 45 high knownGene_ QLYA QLYV ID EQ uc001mmg.3, AQLA AQLA NO: LY hg19_ SMQV SMQV 80 VA knownGene_ uc001mme.3 hg19_ GPRASP1 chrX 101912464 C T 0 LIETL LIETL SEQ LL 47 high knownGene_ LNYPS LNYLS ID NY uc022cbc.1, SRVRT SRVRT NO: LSS hg19_ SF SF 81 RV knownGene_ uc004ejj.4, hg19_ knownGene_ uc022cbd.1, hg19_ knownGene_ uc004eji.4, hg19_ knownGene_ uc010nod.3 hg19_ SLC22A16 chr16 110746270 C T 0 PQLFV PQLFV SEQ GT 50 high knownGene_ GTMA GTMT ID MT uc003pue.3, LLSGV LLSGV NO: LLS hg19_ LTL LTL 82 GV knownGene_ uc003puf.3 hg19_ CNR1 chr6  88853864 G A 0 GKMN GKMN SEQ MV 50 high knownGene_ KLIKT KLIK ID FAF uc011dzr.2, VFAFC MVFA NO: CS hg19_ SML FCSM 83 ML knownGene_ L uc011dzt.2, hg19_ knownGene_ uc010kbz.3, hg19_ knownGene_ uc010kca.3, hg19_ knownGene_ uc003pmq.4, hg19_ knownGene_ uc011dzs.2, hg19_ knownGene_ uc021zco.1 hg19_ TRRAP chr7  98519385 C T 0 AELM AELM SEQ LM 53 high knownGene_ QALW QALW ID QA uc011kis.2, RTLRN CTLR NO: LW hg19_ PADS NPADS 84 CT knownGene_ L uc003upp.3, hg19_ knownGene_ uc003upr.3 hg19_ SARDH chr9 136596596 G A 0 MSLG MSLG SEQ MS 54 high knownGene_ KAYG KVYG ID LG uc011mdo.2 VESH VESH NO: KV VL VL 85 YG V hg19_ SARDH chr9 136596596 G A 0 KRLM KRLM SEQ MS 54 high knownGene_ SLGK SLGK ID LG uc004cep.4, AYGV VYGV NO: KV hg19_ ESHVL ESHVL 86 YG knownGene_ V uc004ceo.3, hg19_ knownGene_ uc011mdn.2 hg19_ SIGLEC5 chr19  52131128 G A 0 GFTCR GFTCR SEQ LL 55 high knownGene_ AQHP AQHL ID GF uc002pxe.4 LGFLQ LGFLQ NO: LQI IFL IFL 87 FL hg19_ CASC3 chr17  38324513 C T 0 PNPGL PNPGL SEQ GL 60 high knownGene_ YPPPV YPPLV ID YPP uc002hue.3, SMSP SMSP NO: LVS hg19_ GQP GQP 88 M knownGene_ uc010cwt.1 hg19_ RAB40B chr17  80616484 C T 0 QAYA QAYA SEQ MT 66 high knownGene_ ERLG ERLG ID FFE uc002kft.3 VTFFE MTFFE NO: VSP VSPL VSPL 89 L hg19_ KCNG1 chr20  49626482 G A 0 FGTIL FGTIL SEQ FLC 73 high knownGene_ TFLRA TFLCA ID AG uc002xwa.4, GKLR GKLR NO: KL hg19_ LLR LLR 90 RL knownGene_ uc002xwb.3 hg19_ CNR1 chr6  88853864 G A 0 GKMN GKMN SEQ KM 79 medium knownGene_ KLIKT KLIK ID NK uc011dzr.2, VFAFC MVFA NO: LIK hg19_ SML FCSM 91 MV knownGene_ L uc011dzt.2, hg19_ knownGene_ uc010kbz,3, hg19_ knownGene_ uc010kca.3, hg19_ knownGene_ uc003pmq.4, hg19_ knownGene_ uc011dzs.2, hg19_ knownGene_ uc021zco.1 hg19_ SLC22A16 chr6 110746270 C T 0 PQLFV PQLFV SEQ QL 84 high knownGene_ GTMA GTMT ID FV uc003pue.3, LLSGV LLSGV NO: GT hg19_ LTL LTL 92 MT knownGene_ L uc003puf.3 hg19_ PIK3CA chr3 178916891 G A 0 ETTRL ETRRL SEQ QL 84 high knownGene_ CDLR CDLQ ID FQP uc003fjk.3 LFQPF LFQPF NO: FL LKV LKV 93 KV hg19_ CASC3 chr17  38324639 C T 0 YAPG YAPG SEQ YA 93 high knownGene_ LPPP ALPPL ID PG uc002hue.3, PPPHL PPPHL NO: AL hg19_ YPN YPN 94 PPL knownGene_ uc010cwt.1 hg19_ SLC6A20 chr3  45817323 G A 0 NGGV NGGV SEQ VQ 95 high knownGene_ QWEP QWEP ID WE uc011bai.2, ALCLL VLCLL NO: PV hg19_ LAWL LAWL 95 L knownGene_ uc011baj.2 hg19_ ABCA10 chr17  67178331 G A 0 ALMG ALMG SEQ GIF 105 high knownGene_ IFNFT IFNFM ID NF uc010dfa.1, ELIQM ELIQM NO: ME hg19_ EST EST 96 LI knownGene_ uc010dfb.1 hg19_ CD163L1 chr12   7531888 G A 0 VGVIC VGVIC SEQ AL 120 high knownGene_ SDAS SDAL ID DM uc001qsy.3, DMEL DMEL NO: EL hg19_ RLVG RLVG 97 RL knownGene_ V uc010sge.2 hg19_ TMTC4 chr13 101277794 G A 0 PDCY PDCY SEQ CL 122 high knownGene_ YNLG YNLG ID VS uc001vow.1, PLVSA CLVSA NO: AG hg19_ GCPV GCPV 98 CP knownGene_ V uc001vov.1 hg19_ PGM5 chr9  70993145 A G 0 RLIIG RLIIG SEQ RLI 124 high knownGene_ QNGIL QNGV ID IGQ uc004agr.3 STPAV LSTPA NO: NG SC VSC 99 V hg19_ NALCN chr13 102029355 C T 0 QMSP QMSP SEQ GM 134 medium knownGene_ WGML WGML ID LQI uc001vpa.2, RIPRP QIPRP NO: PRP hg19_ LIMI LIMI 100 L knownGene_ uc001voz.2, hg19_ knownGene_ uc001vox.1 hg19_ BTBD11 chr12 108004005 C T 0 FCASR FCASR SEQ KL 148 high knownGene_ KLDA KLDV ID DV uc001tml.1, VAIEA VAIEA NO: VAI hg19_ KFK KFK 101 EA uc001tmk.1, hg19_ knownGene_ uc001tmj.3, hg19_ knownGene_ uc009zut.1 hg19_ SARDH chr9 136596596 G A 0 MSLG MSLG SEQ KV 148 high knownGene_ KAYG KVYG ID YG uc011mdo.2 VESH VESH NO: VE VESH VESH 102 SH VL VL V hg19_ SARDH chr9 136596596 G A 0 KRLM KRLM SEQ KV 148 high knownGene_ SLGK SLGK ID YG uc004cep.4, AYGV VYGV NO: VE hg19_ ESHVL ESHVL 103 SH knownGene_ V uc004ceo.3, hg19_ knownGene_ uc011mdn.2 hg19_ LPA chr6 161006078 G A 0 RIPLY RIPLY SEQ RIP 153 high knownGene_ YPNA YPNV ID LY uc003qtl.3 GLTRN GLTRN NO: YP YCR YCR 104 NV hg19_ ITGB1 chr10  33209310 G T 0 LIIDA LIIDA SEQ LII 164 medium knownGene_ YNSLS YNSIS ID DA uc00liwr.4, SEVIL SEVIL NO: YN hg19_ EN EN 105 SI knownGene_ uc001iwt.4, hg19_ knownGene_ uc001iws.4 hg19_ EEFSEC chr3 127965789 G A 0 QIACQ QIACQ SEQ MV 170 high knownGene_ KLVV KLVM ID LN uc003eki.3 VLNKI VLNKI NO: KID DLL DLL 106 LL hg19_ STK36 chr2 219558685 G A 0 YFLSL YFLSL SEQ LL 172 high knownGene_ LVFRL LVFQL ID VF uc002viv.3, QNLP QNLP NO: QL hg19_ CGM CGM 107 QN knownGene_ L uc002viu.3 hg19_ SLC6A20 chr3  45817323 G A 0 NGGV NGGV SEQ VL 173 high knownGene_ QWEP QWEP ID CL uc011bai.2, ALCLL VLCLL NO: LL hg19_ LAWL LAWL 108 L knownGene_ uc011baj.2 hg19_ C6 chr5  41149516 T G 0 DYFTS DYFTS SEQ FTS 212 high knownGene_ PACKF PACTF ID PA uc003jml.2, LAEK LAEK NO: CTF hg19_ CLN CLN 109 L knownGene_ uc003jmk.3 hg19_ EEFSEC chr3 127965789 G A 0 QIACQ QIACQ SEQ KL 214 medium knownGene_ KLVV KLVM ID VM uc003eki3 VLNKI VLNKI NO: VL DLL DLL 110 NKI hg19_ SLC32A1 chr20  37357184 G A 0 LWHQ LWHQ SEQ VFF 225 high knownGene_ VFFD VFFDI ID DIA uc002xjc.3 VAIFV AIFVI NO: IFV IGGI GGI 111 hg19_ NEFM chr8  24771944 C T 0 ALRK ALRK SEQ AL 256 high knownGene_ DIEEA DIEEV ID RK uc003xed.4, SLVKV SLVKV NO: DIE hg19_ ELD ELD 112 EV knownGene_ uc011lac.1 hg19_ ACTN2 chr1 236902618 G A 0 ASELL ASELL SEQ ELL 262 medium knownGene_ EWIRR EWIH ID EW uc001hyf.2, TIPWL RTIPW NO: IHR hg19_ EN LEN 113 T knownGene_ uc001hyg.2, hg19_ knownGene_ uc009xgi.1 hg19_ NEDD9 chr6  11185718 C T 0 ISLLN ISLLN SEQ AID 272 medium knownGene_ AIDAL AIDTL ID TLF uc010joz.2, FSCVS FSCVS NO: SC hg19_ SA SA 114 V knownGene_ uc031sms1, hg19_ knownGene_ uc003mzv.2, hg19_ knownGene_ uc003mzw.3 hg19_ POU6F2 chr7  39503921 C T 0 KLDIT KLDIT SEQ KL 274 medium knownGene_ PKSA PKSV ID DIT uc003thb.2, QKIKP QKIKP NO: PKS hg19_ VLE VLE 115 V knownGene_ c022acb.1 hg19_ SLC9A1 chr1  27436202 C T 0 VGIVD VGIVD SEQ IVD 284 high knownGene_ IFLGF IFLSFL ID IFL uc001bnm.4, LSFFV SFFVV NO: SFL hg19_ VA A 116 knownGene_ uc001bnn.3 hg19_ SLC13A2 chr17  26817568 G A 0 HWNL HWNL SEQ ITL 304 high knownGene_ HKRIA HKRIT ID RV uc002hbi.3, LRVLL LRVLL NO: LLI hg19_ IVG IVG 117 V knownGene_ uc002hbh.3, hg19_ knownGene_ uc010wal.1, hg19_ knownGene_ uc010wam.2 hg19_ knownGene_ uc010wan.2 hg19_ CNR1 chr6  88853864 G A 0 GKMN GKMN SEQ KLI 312 medium knownGene_ KLIKT KLIK ID KM uc011dzr.2, VFAFC MVFA NO: VF hg19_ SML FCSM 118 AF knownGene_ L uc011dzt.2, hg19_ knownGene_ uc010kbz.3, hg19_ knownGene_ uc010kca.3, hg19_ knownGene_ uc003pmq.4, hg19_ knownGene_ uc011dzs.2, hg19_ knownGene_ uc021zco.1 hg19_ CNR1 chr6  88853864 G A 0 GKMN GKMN SEQ KM 313 high knownGene_ KLIKT KLIK ID VF uc011dzr.2, VFAFC MNFA NO: AF hg19_ SML FCSM 119 CS knownGene_ L M uc011dzt.2, hg19_ knownGene_ uc010kbz.3, hg19_ knownGene_ uc010kca.3, hg19_ knownGene_ uc003pmq.4, hg19_ knownGene_ uc011dzs.2, hg19_ knownGene_ uc021zco.1 hg19_ EDNRB chr13  78492668 G A 0 SLCGR SLCGR SEQ SLC 325 high knownGene_ ALVAL ALVV ID GR vkp.1, VLAC LVLAC NO: AL hg19_ GLS GLS 120 VV knownGene_ uc001vkq.1, hg19_ knownGene_ uc001vko.2, hg19_ knownGene_ uc010aez.1 hg19_ TM2D3 chr15 102182749 G A 0 SFGGL SFGGL SEQ MLI 343 medium knownGene_ GIWTL GIWM ID DV uc002bxi.3, IDVLL LIDVL NO: LLI hg19_ IG LIG 121 G knownGene_ uc002bxh.3, hg19_ knownGene_ uc002bxj.3

TABLE 6 Neoepitopes (10-mer) with high binding affinity for HLA-A0201 expr Score transcript gene chr pos ref alt (RPKM) WT_AA MT_AA MTpep MT_ic50 level hg19_ TM2D3 chr15 102182749 G A 0 FSFGG FSFGG SEQ MLID 4 high knownGene_ LGIWT LGIW ID VLLI uc002bxi.3, LIDVL MLID NO: GV hg19_ LIGV VLLIG 122 knownGene_ V uc002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ TBX18 chr6 85446536 G A 0 LGSSP LGSSP SEQ MMD 6 high knownGene_ SGTM SGTM ID RQM uc003pkl.2 TDRQ MDRQ NO: LPPV MLPP MLPP 123 VE VE hg19_ SLC9A1 chr1 27436202 C T 0 HVGIV HVGIV SEQ FLSF 6 high knownGene_ DIFLG DIFLS ID LSFF uc001bnm.4, FLSFF FLSFF NO: VV hg19_ VVAL VVAL 124 knownGene_ uc001bnn.3 hg19_ GPRASP1 chrX 101912464 C T 0 SLIET SLIET SEQ SLIE 10 high knownGene_ LLNYP LLNY ID TLLN uc022cbc.1, SSRVR LSSRV NO: YL hg19_ TSFL RTSFL 125 knownGene_ uc004ejj.4, hg19_ knownGene_ uc022cbd.1, hg19_ knownGene_ uc004eji.4, hg19_ knownGene_ uc010nod.3 hg19_ NOX5 chr15 69329504 G A 0 EKAIG EKAIG SEQ HMA 13 high knownGene_ LAVSR LAVSH ID AVCI uc010bid.2, MAAV MAAV NO: MEV hg19_ CIMEV CIMEV 126 knownGene_ uc002arr.2, hg19_ knownGene_ uc002ars.2, hg19_ knownGene_ uc002arq.2, hg19_ knownGene_ uc010bie.2, hg19_ knownGene_ uc002arp.2 hg19_ GPRASP1 chrX 101912464 C T 0 SLIET SLIET SEQ TLLN 16 high knownGene_ LLNYP LLNY ID YLSS uc022cbc.1, SSRVR LSSRV NO: RV hg19_ TSFL RTSFL 127 knownGene_ uc004ejj.4, hg19_ knownGene_ uc022cbd.1, hg19_ knownGene_ uc004eji.4, hg19_ knownGene_ uc010nod.3 hg19_ SIGLEC5 chr19 52131128 G A 0 GGFT GGFT SEQ HLL 19 high knownGene_ CRAQ CRAQ ID GFL uc002pxe.4 HPLGF HLLGF NO: QIFL high LQIFL LQIFL 128 N N hg19_ BTBD11 chr12 108045467 A C 0 EIMEL EIMEL SEQ LLSA 22 high knownGene_ LSAA LSAAT ID ATFF uc001tml.1, KFFQL FFQLE NO: QL hg19_ EALQ FFQLE 129 knownGene_ R ALQR uc001tmk.1, hg19_ knownGene_ uc0014tmm.1 hg19_ SARDH chr9 136596596 G A 0 YKRL YKRL SEQ LMS 22 high knownGene_ MSLG MSLG ID LGK uc004cep.4, KAYG KVYG NO: VYG hg19_ VESH VESH 130 V knownGene_ VLS VLS uc004ceo.3, hg19_ knownGene_ uc011mdn.2 hg19_ SLC6A15 chr12 85279797 G A 0 ILLMV ILLMV SEQ MVI 28 high knownGene_ IGILF IGIPFF ID GIPF uc001szy.4, FLELS FLELS NO: FFL hg19_ VGQ VGQ 131 knownGene_ uc001szv.4 hg19_ SLC6A15 chr12 85279797 G A 0 MVIGI MVIGI SEQ MVI 28 high knownGene_ PLFFL PFFFL ID GIPF uc010sul.3 ELSVG ELSVG NO: FFL Q Q 132 hg19_ OPRK1 chr8 54142147 C T 0 VLVV VLVV SEQ FVIC 33 high knownGene_ VAVFV VAVFV ID WTPI uc003xrh.1, VCWT ICWTP NO: HI hg19_ PIHIFI IHIFI 133 knownGene_ uc022aup.1, hg19_ knownGene_ uc0101yc.1, hg19_ knownGene_ uc003xri.1 hg19_ FASN chr17 80049217 G A 0 AFLS AFLS SEQ MLN 35 high knownGene_ MLND MLND ID DIVA uc002kdu.3 IAAVP IVAVP NO: VPA ATAM ATAM 134 PF PF hg19_ SLC32A1 chr20 37357184 G A 0 LLWH LLWH SEQ LLW 35 high knownGene_ QVFF QVFF ID HQV uc002xjc.3 DVAIF DIAIF NO: FFDI VIGGI VIGGI 135 C C hg19_ MAP3K1 chr5 56181765 C T 0 LFIEW LFIEW SEQ WMA 37 high knownGene_ MAGG MAGG ID GGL uc003jqw.4 SVAHL LVAHL NO: VAH LSKY LSKY 136 L G G hg19_ WWP2 chr16 69942692 C T 0 WEQR WEQR SEQ ELPN 38 high knownGene_ ELPNG ELPNG ID GCV uc010vlm.2, RVYY CVYY NO: YYV hg19_ VDHN VDHN 137 knwonGene_ TK TK uc002exu.2, hg19_ knownGene_ uc031qwu.1, hg19_ knownGene_ uc002exv.2 hg19_ MYO1D chr17 31087632 C T 0 DAFA DAFA SEQ HLFC 39 high knownGene_ KAIYE KAIYE ID WIV uc002hhp.1, RLFC HLFC NO: TRI hg19_ WIVT WIVT 138 knownGene_ RI RI uc002hho.1, hg19_ knownGene_ uc010wcb.2 hg19_ SLC6A20 chr3 45817323 G A 0 ENGG ENGG SEQ VNC 40 high knownGene_ VQWE VQWE ID LLLA uc011bai.2, PALCL PVLCL NO: WLV hg19_ LLAW LLAW 139 knownGene_ LV LV uc011baj.2 hg19_ LIN7A chr12 81283099 T G 0 LLEKL LLEKL SEQ KLQ 41 medium knownGene_ QESGE QESG ID ESG uc001szj.1 VPVH DVPV NO: DVP KLQSL HKLQ 140 V SL hg19_ HTR3A chr11 113853876 G A 0 VGKS VGKS SEQ YMY 42 high knownGene_ PNIPY PNIPY ID IRHQ uc010rxb.2, VYIRH MYIR NO: GEV hg19_ QGEV HQGE 141 knownGene_ Q VQ uc010rxa.2, hg19_ knownGene_ uc010rxc.2 hg19_ CASC3 ch217 38325584 C T 0 HLYPN HLYPN SEQ HLYP 45 high knownGene_ TQAPS TQAPL ID NTQ uc002hue.3, QVYG QVYG NO: APL hg19_ GVTY GVTY 142 knownGene_ Y Y uc010cwt.1 hg19_ HOXA7 chr7 27194754 G A 0 KEFHF KEFHF SEQ YLM 48 high knownGene_ NRYLT NRYL ID RRR uc003sys.3 RRRRI MRRR NO: RIEI EIAH RIEIA 143 H hg19_ STK36 chr2 219558685 G A 0 LYFLS LYFLS SEQ SLLV 48 high knownGene_ LLVFR LLVFQ ID FQL uc002viv.3, LQNLP LQNLP NO: QNL hg19_ CGME CGME 144 knownGene_ uc002viu.3 hg19_ ABCA10 chr17 67178331 G A 0 NALM NALM SEQ LMGI 56 high knownGene_ GIFNF GIFNF ID FNF uc010dfa.1, TELIQ MELIQ NO: MEL hg19_ MESTS MESTS 145 knownGene_ uc010dfb.1 hg19_ KCNG1 chr20 49626482 G A 0 AFGTI AFGTI SEQ FLCA  60 high knownGene_ LTFLR LTFLC ID GKL uc002xwa.4, AGKL AGKL NO: RLL hg19_ RLLRE RLLRE 146 known_Gene uc002xwb.3 hg19_ TM2D3 chr15 10282749 G A 0 FSFGG FSFGG SEQ GLGI 62 high knownGene_ LGIWT LGIW ID WML uc002bxi3, LIDVL MLID NO: IDV hg19_ LIGV VLLIG 147 knownGene_ V uc002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ TPO chr2 1497732 T C 0 VWLG VWLG SEQ GLA 65 high knownGene_ GLAE GLAE ID ENLL uc002qwx.3, NFLPR NLLPR NO: PRA hg19_ ARTGP ARTGP 148 knownGene_ L L uc002qww.3, hg19_ knownGene_ uc010yio.2, hg19_ knownGene_ uc002qwr.3, hg19_ knownGene_ uc002qwu.3, hg19_ knownGene_ uc010yip.2 hg19_ SLC9A1 chr1 27436202 C T 0 HVGIV HVGIV SEQ IFLS 67 high knownGene_ DIFLG DIFLS ID FLSF uc001bnm.4, FLSFF FLSFF NO: FV hg19_ VVAL VVAL 149 knownGene_ uc001bnn.3 hg19_ ABCA5 chr17 67257396 G A 0 FWSFI FWSFI SEQ VLA 68 high knownGene_ YSVA YSVAV ID CIAI uc002jig.2, ALACI ACIA NO: TEI hg19_ AITEI ITEI 150 knownGene_ uc002jid.2, hg19_ knownGene_ uc002jib.2, hg19_ knownGene_ uc002jic.2, hg19_ knownGene_ uc002jif.2 hg19_ TMTC4 chr13 101277794 G A 0 YPDC YPDC SEQ CLYA 68 high knownGene_ YYNL YYNL ID DLN uc001vot.3, GRLYA GCLYA NO: RHV hg19_ DLNR DLNR 151 knownGene_ HV HV uc010tja.2, hg19_ knownGene_ uc001vou.3 hg19_ FASN chr17 80049217 G A 0 AFLS AFLS SEQ FLS 71 high knownGene_ MLND MLND ID MLN uc002kdu.3 IAAVP IVAVP NO: DIVA ATAM ATAM 152 DIVA PF PF hg19_ SF3A1 chr22 30730630 C T 0 LAYY LAYY SEQ NMA 76 high knownGene_ NMAN NMAN ID NGT uc003ah1.3, GAVIH GTVIH NO: VIHL hg19_ LALK LALK 153 knownGene_ ER ER uc021wnt.1 hg19_ NOX5 chr15 69329504 G A 0 EKAIG EKAIG SEQ GLA 77 high knownGene_ LAVSR LAVSH ID VSH uc010bid.2, MAAV MAAV NO: MAA hg19_ CIMEV CIMEV 154 V knownGene_ uc002arr.2, hg19_ knownGene_ uc002ars.2, hg19_ knownGene_ uc002arq.2, hg19_ knownGene_ uc010bie.2, hg19_ knownGene_ uc002arp.2 hg19_ C2CD3 chr11 73811600 A C 0 QDKL QDKL SEQ KLL 86 high knownGene_ LGLV LGLV ID GLV uc001ouu.2 KLPLH KRPL NO: KRPL QFYM HQFY 155 SF MSF hg19_ SLC32A1 chr20 37357184 G A 0 LLWH LLWH SEQ QVFF 87 high knownGene_ QVFF QVFF ID DIAI uc002xjc.3 DVAIF DIAIF NO: FV VIGGI VIGGI 156 C C hg19_ ACVR1B chr12 52374774 G A 0 GSGLP GSGLP SEQ GLPL 89 high knownGene_ LFVQ LFVQ ID FVQ uc021qya.1, RTVAR HTVA NO: HTV hg19_ TIVLQ RTIVL 157 knownGene_ Q uc001rzn.3 hg19_ knownGene_ uco10snn.2, hg19_ knownGene_ uc001rzm.3, hg19_ knownGene_ uc001rzl.3 hg19_ CLSTN3 chr12 7288865 G A 0 GSLAL GSLAL SEQ ALFP 89 high knownGene_ FPGIR FPGIH ID GIHL uc001qsr.3, LETCD LETCD NO: ET hg19_ EPLW EPLW 158 knownGene_ uc001qss.3 hg19_ GSDMC chr8 130789814 C T 0 MPSM MPSM SEQ SML 89 high knownGene_ LERIS LEHIS ID EHIS uc003ysr.3 KNLV KNLV NO: KNL KEI KEI 159 hg19_ SLC22A16 chr6 110746270 C T 0 IPQLF IPQLF SEQ QLF 95 high knownGene_ VGTM VGTM ID VGT uc003pue.3, ALLSG TLLSG NO: MTL hg19_ VLTLK VLTLK 160 L knownGene_ uc003.puf.3 hg19_ WDR7 chr18 54603098 G A 0 RHAL RHAL SEQ SLIA 97 medium knownGene_ SLIAT SLIAT ID TTRP uc0021gk.1, ARPPA TRPPA NO: PA hg19_ FITTI FITTI 161 knownGene_ uc002lgl.1 hg19_ CD1E chr1 158324361 T G 0 FLKP FLKP SEQ FLKP 98 high knownGene_ WSHG WSHG ID WSH uc001fsd.3, NFSK NVSK NO: GNV hg19_ QELK QELK 162 knownGene_ NLQ NLQ uc010pid.2, hg19_ knownGene_ uc001fsf.3, hg19_ knownGene_ uc001frz.3, hg19_ knownGene_ uc001fsj.3 hg19_ knownGene_ uc001fse.3, hg19_ knownGene_ uc001fsk.3, hg19_ knownGene_ uc001fry.3 hg19_ IRX6 chr16 55363164 C T 0 ALQG ALQG SEQ ALQ 98 high knownGene_ LPLNC LPLNC ID GLPL uc002ehx.3, APCPR VPCPR NO: NCV hg19_ RSEPV RSEPV 163 knownGene_ uc002ehy.3 hg19_ PNLIPRP3 chr10 118236283 A C 0 KHLFE KHLFE SEQ HLFE 99 high knownGene_ DSQN DSQN ID DSQ uc001lcl.4 KLGA TLGA NO: NTL EMVI EMVI 164 NT NT hg19_ ERBB2 chr17 37868208 C T 0 DNYL DNYL SEQ YLST 106 high knownGene_ STDV STDV ID DVG uc010wek.2 GSCTL GFCTL NO: FCT VCPL VCPL 165 HN HN hg19_ ERBB2 chr17 37868208 C T 0 YNYL YNYL SEQ YLST 106 high knownGene_ STDV STDV ID DVG uc002hso.3, GSCTL GFCTL NO: FCT hg19_ VCPL VCPL 166 knownGene_ HN HN uc010cwa.3, hg19_ knownGene_ uc002hsl.3, hg19_ knownGene_ uc002hsn.1, hg19_ knownGene_ uc002hsp.3, hg19_ knownGene_ uc010cwb.3, hg19_ knownGene_ uc002hsm.3 hg19_ CCDC135 chr16 57760055 G A 0 DVAE DVAE SEQ FLVT 107 high knownGene_ RVFLV RVFLV ID EERI uc002emk.3, AEERI TEERI NO: QL hg19_ QLRY QLRY 167 knownGene_ H H uc002emi.3, hg19_ knownGene_ uc002emj.3 hg19_ RAB40B chr17 80616484 C T 0 AQAY AQAY SEQ GMT 108 high knownGene_ AERL AERL ID FFEV uc002kft.3 GVTFF GMTF NO: SPL EVSLP FEVSP 168 C LC hg19_ CLSTN3 chr12 7288865 G A 0 GSLAL GSLAL SEQ SLAL 111 high knownGene_ FPGIR FPGIH ID FPGI uc001qsr.3, LETCD LETCD NO: HL hg19_ EPLW EPLW 169 knownGene_ uc001qss.3 hg19_ SOX6 chr11 16077437 G A 0 SPLQL SPLQL SEQ YVA 115 high knownGene_ QQLY QQLY ID QLA uc001mmd3, AAQL VAQL NO: SMQ hg19_ ASMQ ASMQ 170 V knownGene_ VS VS uc001mmf.3 hg19_ SOX6 chr11 16077437 G A 0 NHKQI NHKQI SEQ YVA 115 high knownGene_ EQLYA EQLY ID QLA uc001mmg.3, AQLA VAQL NO: SMQ hg19_ SMQV ASMQ 171 V knownGene_ S VS uc001mme.3 hg19_ MYO1D chr17 31087632 C T 0 DAFA DAFA SEQ AIYE 118 high knownGene_ KAIYE KAIYE ID HLFC uc002hhp.1, RLFC HLFC NO: WI hg19_ WIVT WIVT 172 knownGene_ RI RI uc002hho.1, hg19_ knownGene_ uc010wcb.2 hg19_ CAMTA1 chr1 7797322 C T 0 HFSCT HFSCT SEQ LMW 120 high knownGene_ PLMW PLMW ID VCA uc010nzv.1, ACAL VCAL NO: LGH hg19_ GHLE GHLE 173 L knownGene_ AA AA uc001aok.4, hg19_ knownGene_ uc001aoi.3, hg19_ knownGene_ uc001aoj.3 hg19_ SLC9A1 chr1 27436202 C T 0 HVGIV HVGIV SEQ GIVD 125 high knownGene_ DIFLG DIFLS ID IFLS uc001bnm.4, FLSFF FLSFF NO: FL hg19_ VVAL VVAL 174 knownGene_ uc001bnn.3 hg19_ CNR1 chr6 88853864 G A 0 FGKM FGKM SEQ KMV 127 high knownGene_ NKLIK NKLIK ID FAFC uc011dzr.2, TVFAF MVFA NO: SML hg19_ CSML FCSM 175 knownGene_ C LC uc011dzt.2, hg19_ knownGene_ uc011dzt.2, hg19_ knownGene_ uc010kbz.3, hg19_ knownGene_ uc010kca.3, hg19_ knownGene_ uc003pmq.4, hg19_ knownGene_ uc011dzs.2, hg19_ knownGene_ uc021zco.1 hg19_ WDR91 chr7 134894422 C T 0 LRDY LRDY SEQ YLE 143 medium knownGene_ WSYL WSYL ID HRLF uc003vsp.2 ERRLF EHRLF NO: SRL SRLED SRLED 176 I I hg19_ ARMC2 chr6 109286202 G T 0 IKKLV IKKLV SEQ YLG 157 high knownGene_ DCLR DCLR ID PTD uc011eao.2, DLGPT YLGPT NO: WQL hg19_ DWQL DWQL 177 A knownGene_ A A uc003pss.4 hg19_ TP53 chr17 7577539 G A 0 NSSC NSSC SEQ GMN 163 high knownGene_ MGGM MGGM ID WRPI uc002gio.3, NRRPI NWRPI NO: LTI hg19_ LTIITL LTIITL 178 knownGene_ uc010cng.2, hg19_ knownGene_ uc002gim.3, hg19_known knownGene_ uc010cni.2, hg19_ knownGene_ uc031qyq.1, hg19_ knownGene_ uc010cnf.2 hg19_ knownGene_ uc002gin.3, hg19_ knownGene_ uc010cnh.2, hg19_ knownGene_ uc002gig.1, hg19_ knownGene_ uc002gih.3, hg19_ knownGene_ uc002gij.3, hg19_ knownGene_ uc002gii.2 hg19_ C6 chr5 41149516 T G 0 NDYF NDYF SEQ FTSP 176 high knownGene_ TSPAC TSPAC ID ACTF uc003jml.2, KFLAE TFLAE NO: LA hg19_ KCLN KCLN 179 knownGene_ N N uc003jmk.3 hg19_ IRAK2 chr3 10264468 C T 0 AAYLP AAYLP SEQ YLPE 179 high knownGene_ EDFIR EDFIW ID DFIW uc003bve.1 VGQL VGQL NO: VG TKRV TKRV 180 D D hg19_ TRRAP chr7 98519385 C T 0 RAEL RAEL SEQ ELM 190 high knownGene_ MQAL MQAL ID QAL uc011kis.2, WRTL WCTL NO: WCT hg19_ RNPA RNPA 181 L knownGene_ DSI DSI uc003upp.3, hg19_ knownGene_ uc003upr.3 hg19_ PTPRC chr1 198711490 A C 0 LRRQ LRRQ SEQ LMV 194 high knownGene_ RCLM RCLM ID HVE uc001gut.2, VQVE VHVE NO: AQYI hg19_ AQYIL AQYIL 182 knownGene_ IH IH uc001gur.2 hg19_known CPQ chr8 97797433 T G 0 NLQQ NLQQ SEQ GLE 194 medium knownGene_ DGLE DGLE ID KGH uc003yhw.3, KVHL KGHL NO: LEPV hg19_ EPVRI EPVRI 183 knownGene_ PH PH uc010mbe.2 hg19_ SLC6A20 chr3 45817323 G A 0 ENGG ENGG SEQ VQW 195 high knownGene_ VQWE VQWE ID EPVL uc011bai.2, PALCL PVCL NO: CLL hg19_ LLAW LLAW 184 knownGene_ LV LV uc011baj.2 hg19_ STK36 chr2 219558685 G A 0 LYFLS LYFLS SEQ YFLS 195 high knownGene_ LLVFR LLVFQ ID LLVF uc002viv.3, LQNLP LQNLP NO: QL hg19_ CGME CGME 185 knownGene_ uc002viu.3 hg19_ NALCN chr13 102029355 C T 0 DQMS DQMS SEQ GML 197 medium knownGene_ PWGM PWGM ID QIPR uc001vpa.2, LRIPR LQIPR NO: PLI hg19_ PLIMI PLIMI 186 knownGene_ R R uc001voz.2, hg19_ knownGene_ uc001vox.1 hg19_ TMEM255B chr13 114469097 C T 0 GLLDP GLLDP SEQ GLL 213 high knownGene_ AEGLS AEGL ID DPAE uc010tkh.2, RRKK LRRK NO: GLL hg19_ TSLWF KTSL 187 knownGene_ WF uc001vuh.3 hg19_ TRRAP chr7 98519385 C T 0 RAEL RAEL SEQ ALW 225 medium knownGene_ MQAL MQAL ID CTLR uc011kis.2, WRTL WCTL NO: NPA hg19_ RNPA RNPA 188 knownGene_ DSI DSI uc003upp.3, hg19_ knownGene_ uc003upr.3 hg19_ CSTF1 chr20 54978601 G A 0 HTED HTED SEQ LLPN 235 high knownGene_ YVLLP YVLLP ID ERTI uc002xxm.1, DERTI NERTI NO: SL hg19_ SLCC SLCC 189 knownGene_ W W uc002xxn.1, hg19_ knownGene_ uc002xxl.1 hg19_ PIK3CA chr3 178916891 G A 0 DETR DETR SEQ LQLF 238 high knownGene_ RLCD RLCD ID QPFL uc003fjk.3 LRLFQ LQLFQ NO: KV PFLKV PFLKV 190 I I hg19_ ADRBK1 chr11 67051736 C T 0 RNFPL RNFPL SEQ TISE 239 high knownGene_ TISER TISEW ID WW uc009yrn.1 WQQE WQQE NO: QQE VAETV VAETV 191 V hg19_ KIF15 chr3 44828026 C T 0 IKKGV IKKGV SEQ FVV 248 high knownGene_ FVVG FVVG ID GVV uc010hiq.3, AVEQ VVEQ NO: EQV hg19_ VVTS VVTS 192 V knownGene_ AA AA uc003cnx.4 hg19_ HS3ST2 chr16 22926539 G A 0 WNAI WNAI SEQ YML 253 high knownGene_ RIGM RIGM ID HLES uc002dli.3 YVLH YMLH NO: WLQ LESW LESW 193 LQY LQY hg19_ CNGA4 chr11 6265440 A C 0 DQQL DQQL SEQ QLD 254 medium knownGene_ DDLQ DDLQ ID DLQ uc001mco.3 TKFAR TTFAR NO: TTFA LLAEL LLAEL 194 E E hg19_ KRAS chr12 25398285 C T 0 EYKL EYKL SEQ KLV 256 high knownGene_ VVVG VVVG ID VVG uc001rgq.1, AGGV ASGV NO: ASG hg19_ GKSA GKSA 195 V knownGene_ LTI LTI uc001rgp.1 hg19_ CASC3 chr17 38324639 C T 0 PYAPG PYAPG SEQ ALPP 260 medium knownGene_ ALPPP ALPPL ID LPPP uc002hue.3, PPPHL PPPHL NO: HL hg19_ YPNT YPNT 196 knownGene_ uc010cwt.1 hg19_ NALCN chr13 102029355 C T 0 DQMS DQMS SEQ QMS 266 medium knownGene_ PWGM PWGM ID PWG uc001vpa.2, LRIPR LQIPR NO: MLQI hg19_ PLIMI PLIMI 197 knownGene_ R R uc001voz.2, hg19_ knownGene_ uc001vox.1 hg19_ SLC6A15 chr12 85279797 G A 0 ILLMV ILLMV SEQ LLM 267 high knownGene_ IGIPLF IGIPFF ID VIGI uc001szy.4, FLELS FLELS NO: PFF hg19_ VGQ VGQ 198 knownGene_ uc001szv.4 hg19_ ACO1 chr9 32418455 G A 0 DGYY DGYY SEQ SLM 286 medium knownGene_ YPDSL YPDSL ID GTD uc003zqw.4, VGTD MGTD NO: SHTT hg19_ SHTT SHTT 199 knownGene_ MI MI uc003zqx.4, hg19_ knownGene_ uc010mjh.1 hg19_ IVL chr1 152882716 A C 0 VKRD VKRD SEQ GMT 295 medium knownGene_ EQLG EQLG ID KEQ uc021ozl.1, MKKE MTKE NO: LLEL hg19_ QLLEL QLLEL 200 knownGene_ PE PE uc001fau.3 hg19_ EDNRB chr13 78492668 G A 0 PSLCG PSLCG SEQ SLCG 309 medium knownGene_ RALVA RALV ID RAL uc001vkp.1, LVLAC VLVL NO: VVL hg19_ GLSR ACGL 201 knownGene_ SR vkq.1, hg19_ knownGene_ uc001vko.2, hg19_ knownGene_ uc010aez.1 hg19_ STK36 chr2 219558685 G A 0 LYFLS LYFLS SEQ FQL 315 high knownGene_ LLVFR LLVFQ ID QNL uc002viv.3, LQNLP LQNLP NO: PCG hg19_ CGME CGME 202 M knownGene_ uc002viu.3 hg19_ TM2D3 chr15 102182749 G A 0 FSFGG FSFGG SEQ GIW 330 medium knownGene_ LGIWT LGIW ID MLID uc002bxi.3, LIDVL MLID NO: VLL hg19_ LIGV VLLIG 203 knownGene_ V uc002bxh.3, hg19_ knownGene_ uc002bxj.3 hg19_ KCNS2 chr8 99441361 C T 0 GYGD GYGD SEQ TMA 345 medium knownGene_ VVPG VVPG ID GKL uc022azb.1, TTAG TMAG NO: TASA hg19_ KLTAS KLTAS 204 knownGene_ AC AC uc003yin.3 hg19_ GNPDA1 chr5 141384531 G A 0 LTKVP LTKVP SEQ ALM 363 high knownGene_ TMAL TMAL ID VGV uc003lmh.4, TVGV MVGV NO: GTV hg19_ GTVM GTVM 205 M knownGene_ DA DA uc010jgh.3, hg19_ knownGene_ uc003lmf.4, hg19_ knownGene_ uc003lmg.4 hg19_ OPRK1 chr8 54142147 C T 0 VLVV VLVV SEQ VLV 372 high knownGene_ VAVFV VAVFV ID VVA uc003xrh.1, VCWT ICWTP NO: VFVI hg19_ PIHIFI IHIFI 206 knownGene_ uc022aup.1, hg19_ knownGene_ uc010lyc.1, hg19_ knownGene_ uc003xri.1 hg19_ SOX6 chr11 16077437 G A 0 SPLQL SPLQL SEQ QLY 384 medium knownGene_ QQLY QQLY ID VAQ uc001mmd.3, AAQL VAQL NO: LAS hg19_ ASMQ ASMQ 207 M knownGene_ VS VS uc001mmf.3 hg19_ SOX6 chr11 16077437 G A 0 NHKQI NHKQI SEQ QLY 384 medium knownGene_ EQLYA EQLY ID VAQ uc001mmg.3, AQLA VAQL NO: LAS hg19_ SMQV ASMQ 208 M knownGene_ S VS uc001mme.3 hg19_ NETO1 chr18 70451000 G A 0 STVAN STVAM SEQ VML 401 medium knownGene_ DVML DVML ID CTG uc002lkw.3, RTGL CTGL NO: LGVI hg19_ GVIR GVIR 209 knownGene_ MW MW uc002lky.2 hg19_ ADCK1 chr14 78397931 G A 0 ISHLL ISHLL SEQ HLL 403 medium knownGene_ NHVP NHVP ID NHV xuj.3, RQML HQML NO: PHQ hg19_ LILKT LILKT 210 M knownGene_ N N uc001xul.3, hg19_ knownGene_ uc001xui.3 hg19_ SLC22A16 chr6 110746270 C T 0 IPQLF IPQLF SEQ MTL 434 medium knownGene_ VGTM VGTM ID LSG uc003pue.3, ALLSG TLLSG NO: VLTL hg19_ VLTLK VLTLK 211 knownGene_ uc003puf.3 hg19_ SLC6A15 chr12 85279797 G A 0 ILLMV ILLMV SEQ LMVI 447 medium knownGene_ IGIPLF IGIPFF ID GIPF uc001szy.4, FLELS FLELS NO: FF hg19_ VGQ VGQ 212 knownGene_ uc001szv.4 hg19_ OPRK1 chr8 54142147 C T 0 VLVV VLVV SEQ AVF 448 medium knownGene_ VAVFV VAVFV ID VIC uc003xrh.1, VCWT ICWTP NO: WTPI hg19_ PIHIFI IHIFI 213 knownGene_ uc022aup.1, hg19_ knownGene_ uc010lyc.1, hg19_ knownGene_ uc003xri.1 hg19_ SLC13A2 chr17 26817568 G A 0 EHWN EHWN SEQ TLRV 457 medium knownGene_ LHKRI LHKRI ID LLIV uc002hbi.3, ALRVL TLRVL NO: GV hg19_ LIVGV LIVGV 214 knownGene_ uc002hbh.3, hg19_ knownGene_ uc010wal.1, hg19_ knownGene_ uc010wam.2, hg19_ knownGene_ uc010wan.2

As shown in Tables 1 to 3 above, the following neoepitopes with high binding affinity for HLA-A2402 were selected through in silico prediction: AYNSISSEVI (SEQ ID NO: 45) (IC₅₀=274 nM), TYQNDNKPEF (SEQ ID NO: 42) (IC₅₀=187 nM), FFYPCHPDVF (SEQ ID NO 49) (IC₅₀=436 nM), VYNMPSTPSF (SEQ ID NO 32) (IC₅₀=38 nM), RYLGPTDWQL (SEQ ID NO 41) (IC₅₀=159 nM), LMVIGIPFFF (SEQ ID NO 48) (IC₅₀=404 nM).

In addition, as shown in Tables 4 to 6, the following neoepitopes with high binding affinity for HLA-A0201 were selected: MLIDVLLIGV (SEQ ID NO: 122) (IC₅₀=4 nM), MMDRQMLPPV (SEQ ID NO: 123) (IC₅₀=6 nM), LMWVCALGHL (SEQ ID NO: 173) (IC₅₀=120 nM), GIVDIFLSFL (SEQ ID NO: 174) (IC₅₀=125 nM), AVFVICWTPI (SEQ ID NO: 213) (IC₅₀=448 nM), TLRVLLIVGV (SEQ ID NO: 214) (IC₅₀=457 nM).

The neoepitopes selected as above were synthesized into prepared MHC-peptide multimers (8-, 9-, 10-mer) for the following experiments. Using these multimers, cells capable of recognizing the same were extracted from patient-derived T cells, and then EBV-negative gastric cancer cell antigen-specific autologous memory T cells were produced.

2. ELISPOT Results for T Cells Activated by Dendritic Cells Loaded with Selected Neoepitope

PBMCs extracted from healthy human blood were separated into monocytes and leukocytes through flow cytometry, and the monocytes were cultured for 2 days in a culture supplemented with cytokines GM-CSF and IL-4 to differentiate into dendritic cells. In addition, the leukocytes were cultured with anti-CD3/CD28 antibody for 3 days, and then cultured in a culture supplemented with cytokine IL-2. The neoepitope peptide selected as above was transferred to the monocyte-differentiated dendritic cells using electroporation. Subsequently, culture was performed for 5 days to identify that the neoepitope has been expressed on the surface of the dendritic cells. Then, the dendritic cells were co-cultured with the leukocytes, which were cultured in a culture supplemented with anti-CD3/CD28 antibody, at a ratio of 1:20 (dendritic cells:leukocytes). In a case of the co-culture, the culture was mixed with a cytokine cocktail containing both cytokine IL-4 that increases the antigen-presenting function of dendritic cells, and cytokines IL-2 and IL-7 that function to help conversion of T cells into memory cells, and culture was performed. After 16 hours, expression levels of IFN-γ in the T cells thus activated were measured with ELISPOT, and the results are illustrated in FIGS. 1 to 4. In order to select memory T cells to which an antigen had been presented through the dendritic cells after co-culture for 72 hours, a magnetic-activated cell sorter (MACS) capable of extracting T cells secreting cytokine IFN-γ was used to extract EBV antigen-specific memory T cells. The extracted memory T cells were cultured in a culture supplemented with cytokines IL-2, IL-7, and IL-15 to maintain their memory function and increase the number of cells, in which the culture was performed until the memory T cells reach the number of cells that can be injected into mice. Here, as a control, cells to which an unstimulated EBV-positive gastric cancer peptide (HLA-A3101) had been delivered were used.

As a result, it was found that the T-cells cultured with dendritic cells loaded with a neoepitope peptide, which has been predicted through Neopepsee, secrete much more IFN-γ than the control regardless of binding affinity of the peptide for HLA.

From these results, it was found that in the present invention, cytotoxic T lymphocytes (CTLs) can be activated by the dendritic cells loaded with each of neoepitopes, which have high binding affinity for HLA-A2402 and HLA-A0201 in Tables 1 to 6 above, and the thus activated T cells have antigen specificity which enables recognition of the neoepitope that is a neoantigen.

Although specific parts of the present invention have been described in detail as above, it is obvious to those skilled in the art that such a specific description is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

The present invention relates to a cancer-specific tumor antigen neoepitope, an antigen-presenting cell loaded with the neoepitope, and a method for activating T cells for cancer treatment using the antigen-presenting cell. 

1.-37. (canceled)
 38. A method for preventing or treating cancer, the method comprising a step of administering to a target individual an antigen-presenting cell loaded with an Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope represented by any one of SEQ ID NOs: 1 to
 214. 39. The method according to claim 38, wherein the antigen-presenting cell is a dendritic cell, a B cell, or a macrophage.
 40. The method according to claim 38, wherein the antigen-presenting cell promotes proliferation or differentiation of T cells.
 41. The method according to claim 38, wherein the Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope exhibits binding affinity with at least one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, β2-microglobulin, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci.
 42. The method according to claim 38, wherein the Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope exhibits binding affinity with at least one of HLA-A*2402 and HLA-A*A0201.
 43. The method according to claim 38, wherein the cancer is Epstein-Barr virus (EBV)-negative cancer.
 44. The method according to claim 38, wherein the cancer is colorectal cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, ovarian cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma.
 45. A method for producing an antigen-presenting cell, the method comprising loading the antigen-presenting cell with an Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope represented by any one of SEQ ID NOs: 1 to
 214. 46. The method according to claim 45, wherein the antigen-presenting cell is a dendritic cell, a B cell, or a macrophage.
 47. The method according to claim 45, wherein the antigen-presenting cell is obtained from peripheral blood mononuclear cells (PBMCs) derived from peripheral blood of a target individual.
 48. The method according to claim 45, wherein the loading is performed by contacting or pulsing the antigen-presenting cell with the cancer-specific tumor antigen neoepitope, wherein the loading is performed by nucleofection of the antigen-presenting cell with an expression vector into which a nucleic acid molecule encoding the cancer-specific tumor antigen neoepitope is inserted, or wherein the loading is performed using a fusion protein that contains the cancer-specific tumor antigen neoepitope and a dendritic cell-specific antibody or a fragment thereof.
 49. A method for preventing or treating Epstein-Barr virus (EBV)-negative cancer, the method comprising a step of administering to a target individual a T cell activated by an antigen-presenting cell loaded with an Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope represented by any one of SEQ ID NOs: 1 to
 214. 50. A method for activating T cells, the method comprising contacting T cells with an antigen-presenting cell loaded with an Epstein-Barr virus (EBV)-negative cancer-specific tumor antigen neoepitope represented by any one of SEQ ID NOs: 1 to
 214. 51. The method according to claim 50, wherein the contacting is performed by co-culture of the T cells with the antigen-presenting cell.
 52. The method according to claim 50, wherein the T cells are obtained from peripheral blood mononuclear cells (PBMCs) of a target individual.
 53. The method according to claim 50, wherein the T cells include one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells.
 54. The method according to claim 51, wherein the co-culture is performed with addition of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), or a combination thereof.
 55. The method according to claim 51, wherein the co-culture is performed with addition of a fusion protein that contains a cytokine and an immunoglobulin heavy chain constant region.
 56. The method according to claim 55, wherein the cytokine is interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), IL-18, tumor necrosis factor (TNF), or granulocyte macrophage colony stimulating factor (GMCSF).
 57. The method according to claim 51, wherein the co-culture is performed with addition of a fusion protein that contains (a) a ligand of CD27, CXCR3, or CD62L and (b) an immunoglobulin heavy chain constant region. 